JP7431802B2 - Compositions and methods for improving library enrichment - Google Patents

Description

CROSS REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/764,753, filed August 15, 2018, under 35 U.S.C. 119(e). The contents of this provisional application are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION Despite rapid advances in next-generation sequencing (NGS) throughput, classical library preparation and enrichment methods are time-consuming and limit throughput. It may be a hindrance. This disclosure describes compositions that include blockers and/or hybridization buffers, methods for using those compositions, and methods for improving the efficiency of nucleic acid selection prior to sequencing.

In one aspect, the present disclosure describes a hybridization buffer that includes a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

In another aspect, the disclosure describes a method that includes using a hybridization buffer. The disclosure also describes kits that include hybridization buffers.

In a further aspect, the disclosure describes a blocker comprising an oligonucleotide, which blocker can be attached to an adapter. The adapter includes a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI). The index region of the adapter and/or the region of the blocker capable of binding to the UMI comprises at least three thymine bases or universal bases and at least one non-universal base.

In yet another aspect, the present disclosure describes blockers that include two oligonucleotides that are not connected. The blocker can bind to at least a portion of the adapter, which includes a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI). Unconnected oligonucleotides contain bases that do not correspond to the index region and/or UMI of the adapter.

The present disclosure also describes methods comprising using the blockers described herein and kits comprising the blockers described herein.

In a further aspect, the disclosure describes a method comprising contacting a library with a blocker in the presence of a hybridization buffer; and contacting the library with a probe, wherein the probe is a member of the library. hybridize to the desired area within. The method does not include the step of amplifying the library members using PCR before sequencing the library members.

As used herein, the term "nucleic acid" is intended to be consistent with usage in the art and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence-specific manner or can be used as templates for the replication of specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. The structure of the analog may have other backbone linkages, including any of a variety known in the art. Naturally occurring nucleic acids typically have deoxyribose sugars (eg, as found in deoxyribonucleic acid (DNA)) or ribose sugars (eg, as found in ribonucleic acid (RNA)). Nucleic acids can include any of the various analogs of these sugar moieties known in the art. Nucleic acids may contain natural or non-natural bases. In this regard, the natural deoxyribonucleic acid may have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and the ribonucleic acid may have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. may have one or more bases selected from the following. Useful non-natural bases that can be included in nucleic acids are known in the art. The term "target" when used with reference to a nucleic acid is intended as a semantic identifier for that nucleic acid in the context of the methods or compositions presented herein, and beyond what is explicitly indicated otherwise. It does not necessarily limit the structure or function of the nucleic acid.

As used herein, the term "T _m " refers to the temperature at which half of the DNA strands of a sample are in a double helix state and half of the DNA strands of a sample are in a random coil state.

As used herein, the term "adapter" and its derivatives (e.g., universal adapter, non-targeting adapter, etc.) generally refers to any linear single strand that can be ligated to a nucleic acid molecule of the present disclosure. Refers to oligonucleotides. In some embodiments, the adapter is not substantially complementary to the 3' or 5' ends of any target sequences present in the sample. In some embodiments, suitable adapter lengths range from 10-100 nucleotides, 12-60 nucleotides, and 15-50 nucleotides in length. Generally, adapters may include any combination of nucleotides and/or nucleic acids. In some embodiments, the adapter may include one or more cleavable groups at one or more positions. In another aspect, the adapter may include a sequence that is substantially identical or substantially complementary to at least a portion of a primer, eg, a universal primer. In some embodiments, the adapter may include an index or tag to aid downstream error correction, identification or sequencing.

As used herein, the term "universal sequence" refers to a region of sequence common to two or more nucleic acid molecules, e.g., adapter-target adapter molecules, where those molecules also have sequence regions that are different from each other. A population of universal capture nucleic acids that are complementary to a portion of the universal sequence, e.g., a universal extension primer binding site, can be used to capture multiple different nucleic acids due to the presence of the universal sequence on different members of the population of molecules. may become possible. Non-limiting examples of universal extension primer binding sites include sequences that are identical or complementary to the P5 and P7 primers. Similarly, the presence of a universal sequence on different members of a population of molecules allows a population of universal primers that are complementary to a portion of the universal sequence, e.g., a universal primer binding site, to replicate or replicate multiple different nucleic acids. It may be possible to amplify. Thus, a universal capture nucleic acid or universal primer comprises a sequence that can specifically hybridize to a universal sequence. Target nucleic acid molecules can be modified to attach universal adapters, such as those described herein, to one or both ends of various target sequences.

The terms "P5" and "P7" may be used to refer to amplification primers, eg, universal primer extension primers. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complementary strands of P5 and P7, respectively. It is understood that any suitable amplification primer can be used in the methods presented herein, and that the use of P5 and P7 are merely exemplary embodiments. The use of amplification primers such as P5 and P7 on flow cells is well known in the art, as exemplified by the disclosures of WO2007/010251, WO2006/064199, WO2005/065814, WO2015/106941, WO1998/044151 and WO2000/018957. It is publicly known. For example, any suitable forward amplification primer, whether immobilized or in solution, can be used for hybridization to complementary sequences and for amplification of the sequences presented herein. can be useful in Similarly, any suitable reverse amplification primer, whether immobilized or in solution, is also provided herein for hybridization to complementary sequences and amplification of sequences. may be useful in methods. Those skilled in the art will understand how to design and use primer sequences suitable for capture and amplification of nucleic acids as provided herein.

As used herein, "amplify," "amplify," or "amplification reaction" and derivatives thereof generally mean that at least a portion of a nucleic acid molecule has at least one additional Refers to any act or process that is replicated or copied into a nucleic acid molecule. The additional nucleic acid molecule optionally comprises a sequence that is substantially identical or substantially complementary to at least a portion of the template nucleic acid molecule. A template nucleic acid molecule can be single-stranded or double-stranded, and additional nucleic acid molecules can independently be single-stranded or double-stranded. Amplification optionally involves linear or exponential replication of the nucleic acid molecule. In some embodiments, such amplification may be performed using isothermal conditions, and in other embodiments, such amplification may include thermocycling. In some embodiments, the amplification is multiplex amplification, which involves amplifying multiple target sequences simultaneously in one amplification reaction. In some embodiments, "amplifying" includes amplifying at least a portion of DNA-based and RNA-based nucleic acids, alone or in combination. Amplification reactions can include any amplification process known to those skilled in the art. In some embodiments, the amplification reaction comprises polymerase chain reaction (PCR).

As used herein, "amplification conditions" and derivatives thereof generally refer to conditions suitable for the amplification of one or more nucleic acid sequences. Such amplification may be linear or exponential. In some embodiments, amplification conditions can include isothermal conditions or can include thermocycling conditions or a combination of isothermal and thermocycling conditions. In some embodiments, conditions suitable for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions are sufficient to amplify a nucleic acid, such as one or more target sequences, or an amplified target sequence ligated to one or more adapters, e.g. Refers to a reaction mixture sufficient to amplify the ligated amplified target sequence. Typically, amplification conditions include a catalyst for amplification or nucleic acid synthesis, e.g., a polymerase; a primer with some degree of complementarity to the nucleic acid to be amplified; and a nucleotide, e.g., to facilitate extension of the primer hybridized to the nucleic acid. These include deoxyribonucleotide triphosphates (dNTPs). Amplification conditions may require hybridization or annealing of the primer to the nucleic acid, extension of the primer, and a denaturation step to separate the extended primer from the nucleic acid sequence being amplified. Typically, but not necessarily, amplification conditions may include thermocycling, and in some embodiments, amplification conditions include multiple cycles of annealing, extension, and separation. The process is repeated. Typically, amplification conditions include cations such as Mg ²⁺ or Mn ²⁺ and may also include various modulators of ionic strength.

As used herein, the term "polymerase chain reaction" ("PCR") refers to a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. Refers to methods such as those described in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202. This process for amplifying a polynucleotide of interest involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycles in the presence of a DNA polymerase. It consists of things. The two primers are complementary to each strand of the double-stranded polynucleotide of interest. First, the mixture is denatured at high temperature, and then the primers are annealed to complementary sequences within the polynucleotide of the molecule of interest. After annealing, the primers are extended by a polymerase to form a new pair of complementary strands. By repeating the steps of denaturation, primer annealing, and polymerase extension many times (termed thermocycling), an amplified segment with a high concentration of the desired polynucleotide of interest is obtained. The length of the desired amplified segment (amplicon) of a polynucleotide of interest is determined by the relative position of the primers to each other, so this length is a controllable parameter. Because this process is repeated, this method is referred to as "polymerase chain reaction" (hereinafter "PCR"). The desired amplified segment of the polynucleotide of interest is said to be "PCR amplified" because it becomes the predominant nucleic acid sequence (in terms of concentration) in the mixture. In a variation of the method discussed above, a target nucleic acid molecule is PCR amplified using a plurality of different primer pairs, in some cases one or more primer pairs per target nucleic acid molecule of interest; Thereby, multiplex PCR reactions can be formed.

As defined herein, "multiplex amplification" refers to the simultaneous amplification of two or more target sequences in a sample. In some embodiments, multiplex amplification is performed such that some or all of the target sequence is amplified in one reaction vessel. The "plexy" or "plex" of a given multiplex amplification generally refers to the number of different target-specific sequences that are amplified during one multiplex amplification. The amplified target sequence can be analyzed by several different methods (e.g. gel electrophoresis followed by densitometry, quantification by bioanalyzer or quantitative PCR, hybridization with labeled probes; incorporation of biotinylated primers followed by avidin-enzyme Detection of the conjugate; incorporation of 32P-labeled deoxynucleotide triphosphates into the amplified target sequence) can also be performed.

As used herein, the term "primer" and its derivatives generally refer to any polynucleotide capable of hybridizing to a target sequence of interest. Typically, a primer serves as a substrate on which nucleotides can be polymerized by a polymerase; however, in some embodiments, a primer can become incorporated into a synthesized nucleic acid strand and the synthesized nucleic acid molecule may provide a site to which another primer can hybridize to stimulate the synthesis of a new strand complementary to the strand. A primer can contain any combination of nucleotides or analogs thereof. In some embodiments, the primer is a single-stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to a multimeric form of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof or their may include mixtures. These terms include analogs of DNA or RNA made of nucleotide analogs as equivalents, and should be construed as applicable to single-stranded (e.g., sense or antisense) and double-stranded polynucleotides. . The term, as used herein, also encompasses cDNA, which is complementary or copy DNA produced from an RNA template, eg, by the action of reverse transcriptase. This term refers only to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded and single-stranded ribonucleic acid ("RNA"). .

As used herein, the terms "ligate", "ligation" and their derivatives generally refer to processes for covalently joining two or more molecules, e.g. Refers to the process of covalently linking multiple nucleic acid molecules to each other. In some embodiments, ligation involves joining nicks between adjacent nucleotides of the nucleic acid. In some embodiments, ligation involves forming a covalent bond between an end of a first nucleic acid molecule and an end of a second nucleic acid molecule. In some embodiments, ligation includes forming a covalent bond between a 5' phosphate group of one nucleic acid and a 3' hydroxyl group of a second nucleic acid to form a ligated nucleic acid molecule. may be included. In some embodiments, a target sequence can be ligated to an adapter to generate an adapter-target sequence. Those skilled in the art will recognize that ligation reactions may not connect all molecules present in the reaction.

As used herein, "ligase" and its derivatives generally refer to any agent that can catalyze the ligation of two substrate molecules. In some embodiments, ligases include enzymes that can catalyze the ligation of nicks between adjacent nucleotides of a nucleic acid. In some embodiments, the ligase is capable of catalyzing the formation of a covalent bond between the 5' phosphate of one nucleic acid molecule and the 3' hydroxyl of another nucleic acid molecule, thereby Enzymes that are capable of forming nucleic acid molecules are included. Suitable ligases may include, but are not limited to, T4 DNA ligase, T4 RNA ligase, thermostable T4 DNA ligase, and E. coli DNA ligase.

The term "flow cell" as used herein refers to a chamber with a solid surface through which one or more fluid reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can be readily used in the methods of the present disclosure include, for example, Bentley et al., Nature 456:53-59 (2008); WO04/018497; WO91/06678; WO07/123744; U.S. Patent No. 7,057,026; U.S. Patent No. 7,211,414; U.S. Patent No. 7,315,019; U.S. Patent No. 7,329,492; U.S. Patent No. 7,405,281; and US Patent Application Publication No. 2008/0108082.

As used herein, the term "library" refers to a collection of members. In one embodiment, the library comprises a collection of nucleic acid members, eg, a collection of whole genome fragments, subgenomic fragments, cDNAs, cDNA fragments, RNA, RNA fragments, or combinations thereof. In some embodiments, some or all of the library members include non-target adapter sequences. Adapter sequences may be located at one or both ends. Adapter sequences can be used, for example, in sequencing methods (eg, NGS methods), for amplification, reverse transcription or cloning into vectors.

"Member" or "library member" or other similar terms, as used herein, refer to nucleic acid molecules, e.g., DNA, RNA, or combinations thereof, that are members of a library. . Typically, the member is a DNA molecule, eg, genomic DNA or cDNA. The member can be genomic DNA that has been fragmented, eg, sheared or enzymatically prepared. Members include sequences derived from the subject and may also include sequences not derived from the subject, e.g. non-target sequences, e.g. adapter sequences, primer sequences, and/or other sequences allowing identification, e.g. an index. .

As used herein, "index" (also referred to as "index region" or "index adapter") refers to a nucleic acid tag that can be used to identify a sample or source of nucleic acid material. refers to When nucleic acid samples are derived from multiple sources, the nucleic acids in each nucleic acid sample can be tagged with different nucleic acid tags so that the origin of the sample can be identified. Any suitable index or Index sets can be used. In some embodiments, the index is from Illumina, Inc. (San Diego, Calif.), a 6-base Index 1 (i7) sequence, an 8-base Index 1 (i7) sequence, an 8-base Index 2 (i5) sequence, a 10-base Index 1 (i7) sequence, or a 10-base Index 2 (i5) sequence.

As used herein, the term "unique molecular identifier" or "UMI" or "barcode" refers to a molecular tag that can be attached to a nucleic acid molecule. UMIs can be used to correct for subsequent amplification bias by directly counting unique molecular identifiers (UMIs) that, when incorporated into nucleic acid molecules, are sequenced after amplification.

As used herein, the term "tagmentation" refers to tagmentation by a transposome complex containing a transposase enzyme complexed with an adapter containing a double-stranded recognition sequence (hybridized transposon terminal sequence). Refers to modification of DNA. Tagmentation results in simultaneous DNA fragmentation and ligation of adapters to the 5' ends of both strands of the duplex fragment. After purification to remove the transposase enzyme, gap filling and ligation, a fully double-stranded product results. Additional sequences may be added to the ends of the matched fragments, for example, by PCR, ligation, or any other suitable method known to those skilled in the art. See, eg, US Patent Application Publication No. 2017/0044525.

The term "and/or" means one or more of the listed elements, or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain advantages in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

The term "comprises" and variations thereof do not have a limiting meaning when these terms appear in the specification and claims.

Whenever embodiments are described herein using language such as “include,” “includes,” or “including,” “from” It is understood that other similar embodiments described with respect to "consisting essentially of" and/or "consisting essentially of" are also provided. The term "consisting of" is limited to what precedes the phrase "consisting of." That is, "consisting of" indicates that the listed element is essential or essential and that no other elements must be present. The term "consisting essentially of" includes any of the elements listed before the phrase and may include other elements other than those listed, provided that An element indicates that it does not interfere with or contribute to the activity or effect specified in this disclosure for the recited element.

Unless otherwise identified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

As used herein, the term "each" when used in reference to a collection of items is intended to identify an individual term within that collection, but the context does not necessarily refer to all terms in the set, unless clearly indicated otherwise.

The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4 , 5, etc.).

For any method disclosed herein that includes separate steps, those steps can be performed in any practicable order. Also, any combination of two or more steps can be performed simultaneously, as appropriate.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. In the following description, illustrative embodiments are illustrated in more detail. At several places throughout this application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the listed list serves only as a representative group and is not to be construed as an exclusive list.

Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "some embodiments," or the like refer to the specific embodiment described in connection with that embodiment. is meant to be included in at least one embodiment of the present disclosure. Accordingly, the appearance of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment of the disclosure. Moreover, the particular features, arrangements, compositions or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, etc. used in the specification and claims are to be understood as modified in all cases by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired characteristics sought to be obtained by the invention. At a minimum, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should be construed at least in light of the number of significant figures reported and by applying normal rounding techniques. It should be.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless the heading specifically states that it does so.

Although numerical ranges and numerical parameters expressing the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, all numerical values inherently include ranges that necessarily result from the standard deviations found in their respective test measurements.

1A-1B are schematic diagrams illustrating exemplary embodiments of hybrid capture and compared off-target capture during enrichment. Figure 1A. The probe includes a region designed to hybridize to a region of interest within the target genome, and a ligand (eg, a biotin group) that enables subsequent capture of the probe. Once hybridization is complete and a DNA template-probe hybrid has been formed, by using a capture means (i.e., a component that has an affinity for the probe, such as, for example, streptavidin-coated magnetic beads) It binds to the probe hybridized to the DNA target and removes the target from the pool of oligonucleotides. Figure 1B. Unwanted or off-target oligonucleotides may be recovered during hybrid capture due to interactions between terminal adapter sequences in the target sequence and terminal adapter sequences in the off-target library preparation product. . Same as above.

FIG. 2 shows an exemplary mechanism by which a blocker prevents hybridization between adapter sequences and prevents off-target capture.

3A-3I show diagrams of exemplary blockers. Figure 3A. Diagram of a NEXTERA blocker containing bases complementary to the 8 or 10 base index region of the adapter. Figure 3B. Diagram of NEXTERA blockers modified with 8 or 10 deoxyinosine bases in the index region of the adapter and the corresponding region of the blocker. Figure 3C. Diagram of TRUSEQ blockers modified with 6, 8 or 10 deoxyinosine bases in the index region of the adapter and the corresponding region of the blocker. Figure 3D. Diagram of NEXTERA and TRUSEQ blockers containing 6 or 8 thymines in the index region of the adapter and the corresponding region of the blocker. Figure 3E. Diagram of a split NEXTERA blocker comprising a component corresponding to a region 5' to the 8 or 10 base index region and a component corresponding to a region 3' to the index region. These constructs do not contain any bases corresponding to the index region of the adapter (indicated by the 8 or 10 base gap corresponding to the index region). Figure 3F. Diagram of a split TRUSEQ blocker comprising components corresponding to regions 5' to the 8 or 10 base index region and components corresponding to regions 3' to the index region. These constructs do not contain any bases corresponding to the index region of the adapter (indicated by the 8 or 10 base gap corresponding to the index region). Figure 3G. Diagram of NEXTERA blocker corresponding to only part of the adapter region 3' of the index region. Figure 3H. Diagram of a TRUSEQ blocker paired with only a portion of the adapter region 3' of the index region. Figure 3I. Diagram of NEXTERA or TRUSEQ blockers that correspond only to a portion of the adapter region (eg, p5 or p7 sequences) 5' of the index region. Same as above. Same as above.

FIG. 4 is a graph of the Padded Read Enrichment (a measure of the amount of on-target DNA captured) results obtained using blockers as described in Example 2.

Figure 5 shows modified G (+G) or random (A, T, G or C) (N) nucleotides at the 5th position of the portion of the blocker sequence that corresponds to the index sequence, as described in Example 3. shows a diagram of a blocker containing.

6A-6B show blocker constructs and results as described in Example 4. FIG. 6A shows a pair of split blockers comprising (1) a first sequence complementary to a 25-30mer region 5′ of the index region and a second sequence complementary to a 34-35mer region 3′ of the index region. , (2) a blocker that pairs only with the 3′ portion of the adapter (“Inner” blocker), (3) a blocker that pairs only with the 5′ portion of the adapter (“Outer” blocker), and (4) a blocker that pairs with only the 5′ portion of the adapter (“Outer” blocker). A diagram of various blocker constructs including the corresponding short (8 nucleotide) blockers (“Short8nt”) is shown. Figure 6B shows Padded Read Enrichment results achieved using XGEN Blocking Oligo (Integrated DNA Technologies, Coralville, IA), a split blocker, or a blocker that binds only a portion of the adapter.

7A-7F show the results when using an enhanced hybridization buffer containing dextran sulfate, as described in Example 5. Figure 7A shows the results of Padded Read Enrichment achieved using various concentrations of dextran sulfate in the hybridization buffer. Figure 7B shows the Padded Read Enrichment achieved using a 100 μL hybridization volume and four different probe panels and three different buffer conditions (IDT hybridization buffer, Illumina non-enhanced hybridization buffer and Illumina enhanced hybridization buffer). Showing results. FIG. 7C shows Padded Read Enrichment and coverage uniformity results for all four probe panels at various hybridization incubation times with and without an additional temperature gradient step. The black dotted line in the left panel shows the results of hybridization in buffer without dextran sulfate. Three sets of sequencing results including NOVASEQ (Fig. 7D), NEXTSEQ (Fig. 7E) and ISEQ (Fig. 7F) (percent reads (PF) identified for NOVASEQ and NEXTSEQ, and index percentage (%) for ISEQ) )) and the associated coefficient of variation (CV) for runs with dextran sulfate hybridization buffer and a library volume of 30 μL are shown. Same as above. Same as above. Same as above. Same as above.

8A-8C show the results of the hybridization assay described in Example 6. Figure 8A shows the results of Padded Read Enrichment when using modified blockers, increased wash temperatures, and/or crowding agents (dextran sulfate) in the hybridization buffer. Figure 8B shows the results of the IDT exome panel when using the IDT The results of achieved Padded Read Enrichment are shown. Figure 8C shows detection of somatic variants by a single hybridization enrichment protocol using a 12-gene, 535-probe panel to enrich libraries generated from Horizon Discover HD701 quantitative multiplex (QM) DNA standards. It shows. Same as above. Same as above.

9A-9D show schematic diagrams of example workflows. eBBN indicates bead-based tagmentation with transposome complexes bound to beads; H indicates hybridization; C indicates capture; W indicates washing. E indicates elution; PCR indicates PCR amplification; S indicates sequencing; Q indicates quantitation; SpVac indicates Speed Vacuum enrichment. There is. FIG. 9A shows a schematic diagram of an exemplary workflow for hybrid capture showing library preparation using a bead-based tagmentation protocol. Any suitable library preparation method may be used prior to enrichment. Remarkably, only one round of hybridization and capture is involved. FIG. 9B shows a schematic diagram of an exemplary workflow for hybrid acquisition. FIG. 9C shows a schematic diagram of an exemplary workflow for hybrid capture without PCR. FIG. 9D shows a schematic diagram of an exemplary workflow for PCR-free hybrid capture with reduced hybridization time. Same as above.

FIG. 10 shows BN001 and BN002 (unmodified blockers); BX003 and BX007 (G-Clamp modified blockers); BN007 and BN008 (blockers modified with BNA and deoxyinosine); BN005 as described in Example 8. and BN006 (BNA and sequence-specific modification blockers); or BN023, BN024, BN025, and BN026 (LNA-modified split blockers).

FIG. 11 shows that BN001 and BN002 (unmodified blockers) or BN023, BN024, BN025 and BN026 (LNA modified split blocks) when the corresponding adapters contain 8 or 10 nucleotide indexes as described in Example 9. Figure 3 shows the results of Padded Read Enrichment achieved using a blocker.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS The present disclosure describes compositions that include blockers and/or hybridization buffers, and methods for improving the efficiency of nucleic acid selection prior to sequencing, including methods for improving the efficiency of nucleic acid selection prior to sequencing. Methods of using the compositions and methods involving hybrid capture are included.
Enrichment by hybrid capture

A variety of methods can be used to enrich desired sequences from a complex pool of nucleic acids. These methods include polymerase chain reaction (PCR), molecular inversion probes (MIP), or sequence capture by hybridization ("hybrid capture"). For example, Mamanova et al., Nat. Methods 7:111-118 (2010)); US Patent Application Publication No. 2014/0031240; and US Patent Application Publication No. 2017/0114404.

Next generation sequencing (NGS) applications typically use enrichment with hybrid capture methods. The prepared NGS template pool, or library, is heat denatured and mixed with a pool of capture probe oligonucleotides (“probes”). These probes are designed to hybridize to regions of interest within the target genome, are typically 60 to 200 bases in length, and are further modified to contain a ligand that allows the bound probe to be captured later. Qualified. A common capture method incorporates a biotin group onto the probe. As shown in one embodiment in FIG. 1A, after hybridization to form a DNA template-probe hybrid is complete, capture is performed using a component that has affinity only for the probe. For example, streptavidin-coated magnetic beads can be used to bind the biotin moiety of a biotinylated probe hybridized to a desired DNA target from a library. Washing removes unbound nucleic acids and reduces the complexity of the retained material. The retained material is then eluted from the magnetic beads and submitted to an automated sequencing process.

Although DNA hybridization with a probe can be highly specific, unwanted sequences remain in the enriched pool after completion of the hybrid capture method. Most of these undesired sequences are due to undesired interactions between library members that do not have complementarity to the probe and library members that do have complementarity to the probe (i.e., on-target library members). exists due to no hybridization event. Two types of sequences result in unwanted hybridization during hybrid capture methods: (1) highly repetitive DNA elements found in endogenous genomic DNA; and (2) engineered to enter each library member. adapter sequence at the end.

Repetitive endogenous DNA elements (e.g. Alu sequences or long interspersed repeats (LINEs)) present in one DNA fragment in the complex pool are replaced by another similar element present in another unrelated DNA fragment. can be hybridized to. These fragments may originally originate from completely different locations within the genome and become linked during the hybridization process of the hybrid capture method. If one of these DNA fragments is the desired fragment containing the binding site for the probe, the unwanted fragment will also be captured along with the desired fragment. This class of off-target library members can be reduced by adding excess repetitive elements to the hybridization buffer of a hybridization reaction. Most commonly, human Cot-1 DNA (which binds to Alu, LINE and other repeat sites in the target, thereby blocking the ability of the NGS templates to interact with each other) is added to the hybridization buffer.

Off-target (also referred to as non-target) library members may also be captured due to interactions between terminal adapter sequences in individual library members. Typically, library members include sequence segments of the gene of interest, eg, segments for sequencing. If a member is on-target, the sequence of the gene of interest forms a duplex with the capture probe. On-target sequences can include, for example, exons or introns (or fragments thereof), coding or non-coding regions, enhancers, untranslated regions, particular SNPs, and the like. Typically, library members also include one or more non-target sequences. These non-target sequences usually do not include the target sequence of interest, but include, for example, adapters. Because the pool of library members usually contains at least some identical terminal adapter sequences, those adapter sequences are present in very high effective concentrations in the hybridization solution. As a result, as shown in one embodiment in FIG. 1B, the library member containing the off-target sequence can anneal to the captured target sequence via a portion of the added adapter sequence; Off-target sequences are thereby captured along with on-target library members (eg, by concatenation or "daisy chaining" of sequences that are linked together). The annealed target sequence contains the binding site for the probe so that the entire daisy strand can be captured. Thus, capture of a single desired fragment may introduce a large number of undesired fragments, reducing the overall efficiency of enrichment for the desired segment.

In some embodiments, this disclosure describes methods and compositions for minimizing selection of off-target nucleic acids by hybrid capture.
blocker oligonucleotide

In one aspect, the present disclosure provides a "blocker oligonucleotide" (also referred to herein as a "blocker"), and the use of a blocker to bind at least a portion of an adapter sequence (e.g., A method is described to prevent the selection of off-target nucleic acids during hybrid capture by forming a duplex with at least a portion of the nucleic acid. When used, as shown in one embodiment in FIG. 2, binding of the blocker to the adapter sequence may result in off-target library preparation products that may result in unwanted library members being recovered during hybrid capture. interactions between adapter sequences (eg, formation of concatenation).

In some embodiments, at least two blockers may be used, where the first blocker binds to (e.g., forms a duplex with) the first adapter sequence and the second blocker binds to (e.g., forms a duplex with) the first adapter sequence. The blocker binds to (eg, forms a duplex with) the second adapter sequence. In some embodiments, the first adapter sequence can be present at the 5' end of the library member and the second adapter sequence can be present at the 3' end of the library member. In some embodiments, multiple different blockers may be used.

In some embodiments, the blocking oligonucleotide forms a duplex with the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 or at least 200 library members. , the resulting duplex has a _{T m} higher than that of the duplex formed by the background nucleic acid (eg, the complement of the adapter sequence) and the adapter sequence.

In some embodiments, the blocker-adapter complex exhibits a higher T _m than the adapter-adapter complex. Such a high T _m means that the blocker-adapter complex is likely to form before the adapter-adapter complex, which may prevent off-target capture (e.g., daisy chain formation may be prevented). ) means that In some embodiments, the T _m of the blocker-adapter oligonucleotide duplex is at least 1.5°C, at least 2°C, at least 3°C lower than the T _m of the adapter duplex with its exact complement, at least 4°C, at least 5°C, at least 10°C, at least 15°C, at least 20°C or at least 25°C higher. In some embodiments, the T _m of the blocker-adapter oligonucleotide duplex is up to 2°C, up to 3°C, up to 4°C, up to 5°C lower than the T _m of the adapter duplex with its exact complement. °C, up to 10°C, up to 15°C, up to 20°C, up to 25°C or up to 30°C higher.

In some embodiments, the rate of association of the blocking oligonucleotide with the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members is Higher than the rate of association between the ground nucleic acid (for example, the complementary strand of the adapter sequence) and the adapter sequence. In some embodiments, the rate of association is at least 2 times higher, at least 4 times higher, at least 6 times higher, at least 8 times higher, or at least 10 times higher. In some embodiments, the association rate is up to 4 times higher, up to 6 times higher, up to 8 times higher, up to 10 times higher, or up to 12 times higher.

In some embodiments, the rate of dissociation of the blocking oligonucleotide and the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 or at least 200 library members is It is lower than the dissociation rate between the ground nucleic acid (for example, the complementary strand of the adapter sequence) and the adapter sequence. In some embodiments, the dissociation rate is at least 2 times lower, at least 4 times lower, at least 6 times lower, at least 8 times lower, or at least 10 times lower. In some embodiments, the association rate is up to 4 times lower, up to 6 times lower, up to 8 times lower, up to 10 times lower, or up to 12 times lower.

In some embodiments, the blocking oligonucleotide is formed between the blocking oligonucleotide and the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 or at least 200 library members. The duplex is longer than the duplex formed between an adapter sequence and its complementary strand, eg, the duplex formed between the Watson-Crick strands of a double-stranded adapter. In some embodiments, the duplex between the blocking oligonucleotide and the adapter sequence has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15 or at least 20 nucleotides long.

In some embodiments, the blocker preferably includes a modification that increases the blocker's T _m compared to an oligonucleotide without the modification that has the same sequence as the blocker. For example, in some embodiments, the blocker can be a "T _m elevated oligonucleotide," which increases the thermal melting temperature value of a double-stranded nucleic acid containing the oligonucleotide as a hybridization partner to the same and at least one modified group that increases the nucleobase composition of the oligonucleotide as compared to a double-stranded nucleic acid containing as a hybridization partner an oligonucleotide with an unmodified group. "T _m elevated oligonucleotides" are further described in US Patent Application Publication No. 2014/0031240.

In some embodiments, the blocker includes one or more non-naturally occurring nucleotides. In some embodiments, a blocking oligonucleotide having a non-naturally occurring nucleotide and an adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 or at least 200 library members. The duplex formed between the , the reciprocal of the dissociation rate, or T _m ).

In some embodiments, blockers include modified bases. Any suitable modified base may be included in the blocker. In some embodiments, the modification preferably includes a modification that increases the T _m , i.e., a blocker containing a modified base, as compared to the T _m of a blocker-adapter complex with the same sequence but without the modified base. - Includes modifications that increase the T _m of the adapter complex. Such T _m -increasing modifications include, for example, DNA or RNA oligonucleotides modified to capture low GC regions; bridging oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (also called bicyclic nucleic acid or BNA); tricyclic nucleic acid; peptide nucleic acid (PNA); C5 modified pyrimidine base; propynylpyrimidine; morpholino; Phosphoramidites; 5'-pyrene caps; and the like may be mentioned. In some embodiments, the LNA comprises a ribose portion of the nucleotide modified with an additional bridge connecting the 2' oxygen and 4' carbon. Exemplary BNAs include, for example, U.S. Pat. No. 7,399,845, U.S. Pat. No. 7,427,672, U.S. Pat. No. 7,741,457, U.S. Patent No. 8,022,193, U.S. Patent No. 8,268,980, U.S. Patent No. 8,278,425, U.S. Patent No. 8,278,426, U.S. Pat. No. 8,846,637, US Pat. No. 8,846,639 and US Pat. No. 9,546,368. In some embodiments, the BNA includes constrained ethyl nucleic acid from Ionis Pharmaceuticals (Carlsbad, Calif.) or Biosynthesis, Inc. (Lewisville, TX) 2'-O,4'-aminoethylene bridged nucleic acids. In some embodiments, the PNA includes repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In some embodiments, tricyclic nucleic acids include, for example, the tricyclic nucleic acids disclosed in US Pat. No. 9,221,864. Exemplary phosphoramidites include 1-[5'-O-(4,4'-dimethoxytrityl)-β-D-2'-deoxyribofuranosyl]-9-(2-trifluoroacetamidoethoxy)- 1,3-Diaza-2-oxophenoxazine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (AP-dC-CE phosphoramidite), 5'-dimethoxytrityl- Uridine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2'-OMe-U-CE phosphoramidite), 5'-dimethoxytrityl -N-acetyl-5-methyl-cytidine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2'-OMe-5-Me- C-CE phosphoramidite), 5'-dimethoxytrityl-N-acetyl-cytidine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite ( 2'-OMe-Ac-C-CE phosphoramidite) and the like.

In some embodiments, modifications that increase the T _m of the blocker compared to an oligonucleotide having the same sequence as the blocker without the modification can include non-basic modifications. Such modifications may include, for example, minor glove binder (MGB), spermine, G-clamp or Uaq anthraquinone cap.

In some embodiments, the blocking oligonucleotide is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 nucleotides long. In some embodiments, the blocking oligonucleotide is up to 45, up to 50, up to 55, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150 or up to 200 nucleotides in length.

In some embodiments, a blocking oligonucleotide can contain multiple modifications that increase the T _m of the blocker. In some embodiments, the preferred number of modifications is optimal under stringent conditions (0.1 x SSC) of at least about 1.4°C for duplex DNA containing the modifications as one complementary strand. This is the number that brings about an increase in the T _m value (“optimum high temperature T _m value”). In some embodiments, the preferred number of modifications in the T _m elevated blocking oligonucleotide is such that the T _m of the blocker-adapter oligonucleotide duplex is at least greater than the T _m of the adapter duplex with its exact complement. 1.5°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 10°C, at least 15°C, at least 20°C or at least 25°C higher. In some embodiments, the preferred number of modifications in the T _m elevated blocking oligonucleotide is such that the T _m of the blocker-adapter oligonucleotide duplex is at most greater than the T _m of the adapter duplex with its exact complement. The number increases by 2°C, maximum 3°C, maximum 4°C, maximum 5°C, maximum 10°C, maximum 15°C, maximum 20°C, maximum 25°C, or maximum 30°C.

In some embodiments, the blocking oligonucleotide comprises at least 2 percent (%), at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% modification. In some embodiments, the blocking oligonucleotide comprises at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, of the bases in the blocking oligonucleotide. Contains up to 80%, up to 90% or up to 100% modification.

In some embodiments, modifications to the blocker may be included in a particular pattern. For example, modified bases may be modified every second base (see, e.g., Table 2A, BN021 and BN022), every third base (see, e.g., Table 2A, BN035 and BN036), every fourth base, or every fifth base, or They may be included in some combination (see for example Table 2A, BN036). In some embodiments, each base in the blocker can be modified (see, eg, Table 2A, BN027 and BN028). In some embodiments, guanine, which when modified has a higher affinity than other nucleotides, may be preferentially modified. For example, in some embodiments, LNA or BNA types of guanine may be included in the blocker.

In some embodiments, blockers may include terminal modifications. For example, a blocker may contain a 3' terminal group that prevents the blocker's availability to serve as a primer for DNA synthesis. Such 3' end groups include, for example, 3'-dC, 2',3'-ddC (also referred to herein as 3ddc), inverted dT, 3'-spacer C3 (also referred to herein as (also referred to as 3SpC3).

The blocker may bind to (eg, form a duplex with) any suitable adapter sequence, including any portion thereof. As used herein, a portion of a blocker sequence that binds to a portion of an adapter sequence (e.g., forms a duplex with a portion of an adapter sequence) refers to a portion of a blocker sequence that “corresponds to” a portion of an adapter sequence. ” part of the blocker array. In some embodiments, the corresponding sequence can be the exact complement of the adapter sequence. However, in some embodiments, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 bases in the corresponding sequence are complementary There is a possibility that it is not.

In some exemplary embodiments, blockers may be coupled to adapters such as those shown in Table 1. (The adapter sequence is shown in Table 1A and further information regarding the components of the sequence is shown in Table 1B.) In some exemplary embodiments, the blocker is provided in the Illumina Adapter Sequences (support.illumina. com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-seque nces-1000000002694-07.pdf (available on the World Wide Web). Can be combined.

In some embodiments, the blocker is attached to an adapter that includes a universal sequence, such as a universal adapter and/or universal primer sequence. In some embodiments, the universal primer sequence may include P5, P7, P5' and/or P7'. In some embodiments, the universal primer sequence is V2. A14. METS array, V2. B15. METS array, V2. A14. Complementary strand of METS sequence and/or V2. B15. It may contain the complementary strand of the METS sequence. For example, BN001 as shown in Table 2A has P5 and V2. A14. contains the METS sequence; BN002 as shown in Table 2A contains P7 and V2. B15. contains the METS sequence; BN003 as shown in Table 2A contains P5' and V2. A14. Contains the reverse complement of the METS sequence; BN004 as shown in Table 2A contains P7' and V2. B15. Contains the reverse complement of the METS sequence.

For some sequencing applications, it is desirable for library members to include at least one index and/or UMI sequence. Thus, in some embodiments, the blocker is attached to an adapter, and the adapter includes at least one of an index and a UMI. U.S. Patent Application Publication No. 2014/0031240 describes adapters that include an index (referred to as a "barcode domain" in U.S. Patent Application Publication No. 2014/0031240) to "produce blocking oligonucleotides by using three approaches. ``1) synthesis of a series of blockers that perfectly match each adapter, 2) synthesis of a single blocker with an N-mer domain that pairs with the barcode domain of the adapter, or 3. ) Synthesis of a single blocker with a universal base domain that pairs with the barcode domain of the adapter. Since each different index and/or UMI has a unique sequence, a fully complementary blocker as in approach 1 is a sequence that contains an exact match complementarity to each index and/or UMI sequence present. (see Figure 3A). However, if many different indices and/or UMIs are used, the inclusion of blockers with perfect complementarity may require tens or hundreds of unique blockers for multiplexed amplification, i.e., the cost This can be a less effective approach.

In some aspects, the present disclosure provides blockers and methods of using such blockers for adapters that include an index and/or UMI, e.g., blockers that include thymine in the region of the blocker that corresponds to the index and/or UMI (Figure 3D and Example 2); blockers that include a universal base and at least one non-universal base (Figure 5 and Example 3); or blockers that do not include an index region (e.g., "split blockers" or paired with only part of the adapter); 3E-3I, FIG. 6A and Examples 4, 8 and 9).

In some embodiments, the blocker comprises at least one of the sequences in Table 2A, Table 2B, Table 2C, or Table 3. In some embodiments, the sequences in Table 2A can be used according to tagmentation (eg, NEXTERA) library preparation (Illumina, Inc., San Diego, Calif.). In some embodiments, the sequences in Table 2B can be used according to ligation-based library preparation and/or TRUSEQ preparation (Illumina, Inc., San Diego, Calif.). In some embodiments, the sequences in Table 2C can be used according to tagmentation (eg, NEXTERA) library preparation (Illumina, Inc., San Diego, Calif.). In some embodiments, when the sequences in Table 2 include a BNA-modified nucleic acid, the BNA-modified nucleic acid is manufactured by Biosynthesis, Inc. (Lewisville, TX), 2'-O,4'-aminoethylene bridged nucleic acids. In some embodiments, when the sequences of Table 2 include an LNA-modified nucleic acid, the LNA-modified nucleic acid can include an LNA-containing oligonucleotide from Qiagen (Hilden, Germany). Further information regarding the components of the sequences of Table 2A, Table 2B and Table 2C can be found in Table 1B and Table 2D.

In another aspect, the disclosure describes kits that include blocking oligonucleotides. The blocking oligonucleotide may include a blocker as described herein. In some embodiments, the blocking oligonucleotide is a plurality of blocking oligonucleotides. The kit may further include one or more of a hybridization buffer, a probe, a panel of probes, and a flow cell. The hybridization buffer may include a hybridization buffer as described elsewhere in this disclosure. In some embodiments, the probe is as described elsewhere in this disclosure. In some embodiments, the components may be provided in separate containers. For example, a blocking oligonucleotide can be provided in a first container and another component, such as a hybridization buffer, or a probe or panel of probes, can be provided in a different container. Alternatively, the blocking oligonucleotide and hybridization buffer can be provided in a first container and the probe or panel of probes can be provided in a different container.
Table 1A - Exemplary adapter sequences


Table 2A - Exemplary Blocker Sequence



Table 2B - Exemplary Blocker Sequence


Table 2C - Exemplary Blocker Sequence


Blockers containing thymine

In some embodiments, the blocker contains at least three thymines in the index region of the adapter and/or in the region of the blocker that corresponds to the UMI of the adapter (e.g., in the index region of the adapter and/or the region of the blocker that binds to the UMI). bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases.

In some embodiments, the region of the blocker that corresponds to (e.g., binds to) the index region of the adapter and/or the UMI of the adapter is the index region of the adapter and/or the UMI of the adapter. contains the same number of thymine bases as the number of bases in .

In some embodiments, the blocker comprises at least one sequence of Table 2A, Table 2B, Table 2C, or Table 3. Some exemplary embodiments of such blockers are described in Example 2.
Blocker containing a universal base and at least one non-universal base

In some embodiments, the blocker can include a universal base and at least one non-universal base in the region of the blocker that corresponds to the index region. As used herein, "universal base" refers to common bases such as deoxyadenosine (A), deoxythymidine (T), deoxycytidine (C), deoxyguanosine (G) and uridine (U )) is a modified nucleobase that hybridizes to at least two of the following. In some embodiments, a universal base hybridizes to any common base. As used herein, a "non-universal base" refers to a conventional Watson-Crick base pairing interaction, such as deoxyadenosine (A)-deoxythymidine (T) or deoxyguanosine (G)-deoxycytidine ( C)) (including modified nucleobases in some embodiments) that hybridize only with C)).

In some embodiments, the at least one non-universal base can be included every third base of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base may be present at the third and/or fifth position (from the 5' end) of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base may be present in the second and/or fourth position of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base comprises a modified guanine. In some embodiments, the modified guanine may be modified with LNA or modified with BNA. In some embodiments, the at least one non-universal base comprises a random (A, T, G or C) nucleotide.

While not wishing to be bound by theory, by including a modified guanine or random nucleotide in addition to the universal base in the region of the blocker that corresponds to the index region (as shown in one embodiment in FIG. ), the affinity of the blocker for library members is thought to be improved. Some exemplary embodiments of such blockers are described in Example 3.

Universal bases can include any known universal bases. In some embodiments, the universal bases used in the index region include, for example, 2'-deoxyinosine, 2'-deoxynebularine, 3-nitropyrrole 2'-deoxynucleoside, or 5-nitroindole 2'-deoxy Nucleosides may be mentioned. In some embodiments, universal base combinations may be used in the index region.

The modified guanine can be a guanine that has been modified in any suitable manner (including, for example, LNA modified guanine, BNA modified guanine, etc.). In some embodiments, the modified guanine is preferably an LNA modified guanine.

In some embodiments, the blocker comprises at least one sequence of Table 2A, Table 2B, Table 2C, or Table 4.
split blocker

In some embodiments, the blocker can include at least two unconnected oligonucleotides, where the unconnected oligonucleotides include bases that do not correspond to the index region and/or UMI of the adapter (e.g., the adapter or contains bases that only partially correspond to the index region and/or UMI of the adapter (e.g., a portion of the index region and/or UMI of the adapter (contains bases that only bind together). In some embodiments, the blocker comprises four unconnected oligonucleotides.

In some embodiments, at least two unconnected oligonucleotides hybridize to the same strand of the target sequence. In some embodiments, at least a portion of the blocker may correspond to the 5' end of the adapter and at least a portion of the blocker may correspond to the 3' end of the same adapter. In some embodiments, such as when the blocker includes four unconnected oligonucleotides, the two unconnected oligonucleotides may hybridize to the same strand of the first target sequence; Two unconnected oligonucleotides can hybridize to the same strand of a second target sequence. Exemplary embodiments of such blockers are shown in FIGS. 3E-3I and 6A and described in Examples 4, 8, and 9.

In some embodiments, at least a portion of the blocker may correspond to a universal extension primer. In some embodiments, at least a portion of the blocker may correspond to P5, P7, P5' and/or P7'. In some embodiments, at least a portion of the blocker is V2. A14. METS, V2. B15. METS, V2. A14. Complementary strand of METS and/or V2. B15. It can correspond to the complementary strand of METS.

In some embodiments, the blocker may include the same number of bases as the adapter excluding the portion of the adapter that includes the bases of the index region and/or UMI of the adapter. In some embodiments, the blocker is 1 base less, 2 bases less, 3 bases less, 4 bases less than the number of bases that make up part of the adapter excluding bases in the index region and/or UMI of the adapter. It may contain fewer or 5 fewer bases.

In some embodiments, blockers containing more bases may be preferred (eg, when high enrichment performance is desired). In some embodiments, blockers containing fewer bases may be preferred (eg, when blocker cost, synthesis purity, and/or yield are factors).

In some embodiments, up to 5 bases of the blocker, up to 4 bases of the blocker, up to 3 bases of the blocker, up to 2 bases of the blocker, or only 1 base of the blocker pair with the index region of the adapter and/or the UMI of the adapter. The bases of the blocker may not pair with the index region of the adapter and/or the UMI of the adapter.

In some embodiments, the blocker comprises at least one sequence (or a portion of a sequence) of Table 2A, Table 2B, Table 2C, or Table 5. In some embodiments, the blocker comprises at least one of BN023, BN024, BN025, and BN026 of Table 2A. In some embodiments, the blocker comprises BN023 and BN025 of Table 2A. In some embodiments, the blocker comprises BN024 and BN026 of Table 2A.
Hybridization buffer

In another aspect, the disclosure describes hybridization buffers that include crowding agents and methods of using the hybridization buffers. As used herein, "crowding agent" includes compounds that enable, enhance, or promote crowding of molecules. In some embodiments, crowding agents include inert macromolecules used at concentrations that alter DNA-protein interactions. "Hybridization buffer" is defined as any buffer in which nucleotide-probe hybrids can be formed as part of a hybrid capture assay.

In some embodiments, the crowding agent can include at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, betaine, and the like. Dextran and dextran sulfate include high molecular weight dextran (e.g., dextran with a molecular weight of at least 500,000 Da) and/or low molecular weight dextran (e.g., with a molecular weight of up to 10,000 Da, up to 50,000 Da, or up to 100,000 Da). dextran). In some embodiments, the crowding agent preferably comprises dextran sulfate.

In some embodiments, the crowding agent is at least 0.5% weight/volume (w/v), at least 1% (w/v), at least 1.5% (w/v) in the hybridization buffer. , at least 2% (w/v), at least 3% (w/v) or at least 4% (w/v). In some embodiments, the crowding agent is present at up to 1% (w/v), up to 1.5% (w/v), up to 2% (w/v), up to 3% (w/v) in the hybridization buffer. w/v), up to 4% (w/v), up to 5% (w/v), up to 6% (w/v), up to 8% (w/v) or up to 10% (w/v) may be included in amounts.

As described in some embodiments in Example 5 (see, e.g., FIGS. 7D-7F), some In embodiments, reducing hybridization time and/or performing hybridization in a larger volume of hybridization buffer than a hybridization buffer without crowding agents (e.g., increasing the dilution of the target oligonucleotide) ) may become possible. For example, by including dextran sulfate in the hybridization buffer, hybridization times can be reduced from overnight to approximately 80 minutes at a single temperature, or to 60 minutes with an additional temperature gradient step. It can be done.

In some embodiments, inclusion of crowding agents may improve enrichment specificity. For example, one embodiment is shown in Example 5 (see Figure 8A), where the enrichment specificity was increased from 45% to 80-95%. In some embodiments, the improvement in enrichment specificity due to the inclusion of a crowding agent can be more pronounced as the incubation time becomes shorter.

In some embodiments, by including a crowding agent in the hybridization buffer, enrichment reactions can occur more quickly with lower concentrations of DNA (e.g., in a larger relative volume of hybridization buffer). reactions, etc.). Using such an increase may facilitate the elimination of "pooling-by-volume" and/or sample concentration steps. In general, achieving rapid hybridization reactions requires the use of high concentrations of DNA from library preparations and high concentrations of probe, so some embodiments accommodate this. A small amount of hybridization buffer (eg, less than 20 μL) is required. Achieving this low volume requires a sample concentration step after library preparation (resulting in longer overall assay times and higher costs). By including crowding agents in the hybridization buffer, enrichment reactions can be accomplished more quickly at lower DNA and/or probe concentrations (and in some embodiments, larger volumes). In some embodiments, the inclusion of a crowding agent in the hybridization buffer allows multiple library preparation samples to be combined without an enrichment step prior to hybridization. In some embodiments, samples from different library preparations may be pooled based on volume, allowing for multiplex enrichment, as further described in this disclosure. The "plex" or "plex" of a given multiplex enrichment refers to the number of different library preparations that are combined prior to enrichment (eg, hybridization and capture). In some embodiments, the multiplex enrichment plexes can be up to 2 plexes, up to 4 plexes, up to 6 plexes, up to 12 plexes, up to 24 plexes, up to 48 plexes, up to 96 plexes or more. .

In some embodiments, the hybridization buffer comprising a crowding agent further comprises at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker. In some embodiments, components of the hybridization buffer, e.g., Cot-1 DNA and blocker, are separated from components of the hybridization buffer, including destabilizing agents and crowding agents, until the hybridization reaction is performed. maintained in the state.

In some embodiments, the hybridization buffer can contain human Cot-1 DNA in an amount of at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL, or at least 0.3 mg/mL. In some embodiments, the hybridization buffer can contain human Cot-1 DNA in an amount up to 0.1 mg/mL, up to 0.2 mg/mL, up to 0.3 mg/mL, or up to 0.5 mg/mL. In some embodiments, the hybridization buffer may preferably contain human Cot-1 DNA in an amount of 0.2 mg/mL.

In some embodiments, the hybridization buffer may include a destabilizing agent. In some embodiments, the destabilizing agent can include formamide and/or urea. In some embodiments, the hybridization buffer can include at least 1% (v:v), at least 5% (v:v) or at least 10% (v:v) formamide. In some embodiments, the hybridization buffer can contain up to 5% (v:v), up to 10% (v:v) or up to 15% (v:v) formamide. In some embodiments, the hybridization buffer can include at least 1M urea, at least 2M urea, or at least 4M urea. In some embodiments, the hybridization buffer can contain up to 2M urea, up to 4M urea or up to 6M urea. In some embodiments, the hybridization buffer may preferably include 10% formamide.

In some embodiments, the hybridization buffer may include a buffer. Any suitable buffer may be used. In some embodiments, the buffer includes phosphate salts (e.g., including potassium phosphate or sodium phosphate); Tris-HCl; piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); ); Piperazine-N,N'-bis(3-propanesulfonic acid) (PIPPS); 2-(N-morpholino)ethanesulfonic acid (MES); (4-(2-hydroxyethyl)-1-piperazineethanesulfone) acid) (HEPES) and the like. In some embodiments, the hybridization buffer can include at least 40 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM or at least 70 mM phosphate. In some embodiments, the hybridization buffer can include up to 50 mM, up to 55 mM, up to 60 mM, up to 65 mM, up to 70 mM, up to 75 mM, or up to 80 mM phosphate. In some embodiments, the phosphate-containing buffer can include dihydrogen phosphate and/or monohydrogen phosphate. In some embodiments, the phosphate-containing buffer can include KH ₂ PO ₄ -K ₂ HPO. In some embodiments, the hybridization buffer may preferably include 66.6 mM KH ₂ PO ₄ -K ₂ HPO ₄ .

In some embodiments, hybridization buffers can include salts. Any suitable salt may be included. In some embodiments, the salt can be, for example, NaCl or sodium citrate (eg, trisodium citrate). In some embodiments, the hybridization buffer is at least 0.1M, at least 0.2M, at least 0.3M, at least 0.4M, at least 0.5M, at least 0.6M, at least 0.7M, at least 0. 8M, at least 0.9M or at least 1M salt. In some embodiments, the hybridization buffer is up to 0.2M, up to 0.3M, up to 0.4M, up to 0.5M, up to 0.6M, up to 0.7M, up to 0.8M, up to 0. It may contain up to 9M, up to 1M, up to 2M, up to 3M or up to 4M salt. In some embodiments, the hybridization buffer may preferably include 0.8M NaCl.

In some embodiments, the hybridization buffer contains a detergent (e.g., an anionic detergent (e.g., sodium dodecyl sulfate, sulfonate, alcohol sulfate, alkylbenzene sulfonate, phosphate ester, or carboxylic acid) salts), nonionic surfactants (e.g. polyoxyethylene or glycoside surfactants, e.g. Tween®, Triton or Brij surfactants), cationic surfactants (e.g. quaternary ammonium cationic surfactants) or zwitterionic surfactants (such as CHAPS). In some embodiments, the surfactant is an anionic surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is TWEEN® 20, TWEEN® 80, sodium dodecyl sulfate (SDS), and the like. In some embodiments, the surfactant comprises at least 0.001% volume/volume (v/v), at least 0.01% (v/v), at least 0.05% (v/v) of the hybridization buffer. ), at least 0.1% (v/v), at least 1% (v/v) or at least 5% (v/v). In some embodiments, the detergent comprises up to 0.01% (v/v), up to 0.05% (v/v), up to 0.1% (v/v), up to 1% (v/v), up to 5% (v/v) or up to 10% (v/v). In some embodiments, the hybridization buffer may preferably include 0.04% (v/v) TWEEN® 20.

For example, in some embodiments, the hybridization buffer comprises 0.5% to 10% dextran sulfate, 0.05 mg/mL to 0.5 mg/mL human Cot-1 DNA, 1% to 15% (v/v) Formamide, 40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ , 0.1M to 4M NaCl, and 0.001% to 10% (v/v) Tween 20. In some embodiments, the hybridization buffer (eg, in the final reaction) contains 1.5% dextran sulfate, 0.2 mg/mL Cot-1, 10% (v/v) formamide, 66. May contain 6mM KH ₂ PO ₄ -K ₂ HPO ₄ , 0.8M NaCl, and 0.04% (v/v) TWEEN® 20.

In some embodiments, the hybridization buffer may include a blocker. In some embodiments, the blocker is as described elsewhere in this disclosure. In some embodiments, the blocker is present at a concentration of at least 0.001mM, at least 0.005mM, at least 0.01mM, at least 0.05mM or at least 0.1mM. In some embodiments, the blocker is present at a concentration of up to 0.01mM, up to 0.05mM, up to 0.1mM, up to 0.2mM or up to 0.5mM.

In another aspect, the disclosure describes a kit comprising a hybridization buffer, eg, a hybridization buffer as described herein. In certain embodiments, the kit further includes at least one of a blocker and a probe. In embodiments, the components are provided in separate containers. For example, a hybridization buffer can be provided in a first container and another component, such as a blocker or probe, can be provided in a different container. In some embodiments, the blocker and/or probe is as described elsewhere in this disclosure.
hybrid acquisition

In a further aspect, the disclosure describes a method of hybrid capture. In some embodiments, the method includes steps that include hybridizing the probe to a library member and capturing the probe. In some embodiments, the method further includes pooling the library prior to hybridizing the probe. In some embodiments, the method further includes amplifying the captured sequence after capture. In some embodiments, the method may be used in combination with blocker oligonucleotides as described in this disclosure. In some embodiments, the method can include using a hybridization buffer that includes a crowding agent as described in this disclosure.
library pool

In some embodiments, samples from different library preparations are combined prior to hybridization. In some embodiments, each library that is combined preferably includes a sequence that includes an index region that is distinguishable from the index region of a sequence in any other library that is combined.

In some embodiments, samples from different library preparations may be pooled based on volume to allow multiplex enrichment. The "plex" or "plex" of a given multiplex enrichment refers to the number of different library preparations that are combined prior to enrichment (eg, hybridization and capture). In some embodiments, the multiplex enrichment plexes can be up to 2 plexes, up to 4 plexes, up to 6 plexes, up to 12 plexes, up to 24 plexes, up to 48 plexes, up to 96 plexes or more. .

In some embodiments, a sample derived from a library preparation is derived from a tagmentation reaction. See, eg, US Pat. No. 9,574,226. In some embodiments, the sample derived from the library preparation is derived from the NEXTERA library preparation (Illumina, Inc., San Diego, Calif.). In some embodiments, library preparations can be added directly to hybridization reactions.

In some embodiments, the DNA concentration of each sample from the combined library preparations is at least 0.1 ng/μL, at least 0.3 ng/μL, at least 0.4 ng/μL, at least 0.5 ng/μL , at least 1 ng/μL, at least 2 ng/μL, at least 4 ng/μL, at least 6 ng/μL, at least 8 ng/μL, at least 10 ng/μL, at least 12 ng/μL, at least 20 ng/μL, at least 50 ng/μL, at least 60 ng/μL , at least 100 ng/μL or at least 200 ng/μL. In some embodiments, the DNA concentration of each sample from the combined library preparations is up to 0.4 ng/μL, up to 0.5 ng/μL, up to 1 ng/μL, up to 2 ng/μL, up to 4 ng/μL. μL, maximum 6ng/μL, maximum 8ng/μL, maximum 10ng/μL, maximum 12ng/μL, maximum 15ng/μL, maximum 20ng/μL, maximum 50ng/μL, maximum 100ng/μL, maximum 120ng/μL, maximum 200ng/μL μL, up to 300 ng/μL or up to 400 ng/μL. For example, in some embodiments, the DNA concentration of each sample derived from a library preparation can be within the range of 0.3 ng/μL to 400 ng/μL. For example, in some embodiments, the DNA concentration of each sample derived from a library preparation can range from 0.1 ng/μL to 120 ng/μL. For example, in some embodiments, the DNA concentration of a combination of samples derived from a library preparation can be in the range of 0.1 ng/μL to 120 ng/μL (eg, in 100 μL). For example, in some embodiments, the DNA concentration of each sample derived from a library preparation can be in the range of 0.5 ng/μL to 12 ng/μL. In some embodiments, the DNA concentration of the sample combination derived from the library preparation can be in the range of 0.5 ng/μL to 12 ng/μL (eg, in 100 μL). For example, in some embodiments, the DNA concentration of each sample derived from a library preparation can range from 0.5 ng/μL to 120 ng/μL. In some embodiments, the DNA concentration of the sample combination derived from the library preparation can be in the range of 0.5 ng/μL to 120 ng/μL (eg, in 100 μL).

In some embodiments, at least 1 μL, at least 2 μL, at least 3 μL, at least 5 μL, at least 10 μL, at least 15 μL, at least 20 μL, or at least 25 μL of each sample from the library preparation can be combined. In some embodiments, up to 2 μL, up to 3 μL, up to 5 μL, up to 10 μL, up to 15 μL, up to 20 μL, up to 25 μL, up to 30 μL, up to 40 μL, or up to 50 μL of each sample from the library preparation is combined can be done.

In some embodiments, at least 10 ng, at least 15 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 500 ng, at least 1,000 ng, or at least 5,000 ng of DNA from each library preparation is combined. can be done. In some embodiments, up to 15 ng, up to 25 ng, up to 50 ng, up to 100 ng, up to 200 ng, up to 500 ng, up to 1,000 ng, up to 5,000 ng, up to 10,000 ng, or up to 12,000 ng of each library preparation. DNA from different species can be combined. For example, in some embodiments, 10 ng to 12,000 ng of DNA from each library preparation can be combined.
Hybridization of probes

In some embodiments, methods of hybrid capture include contacting a library with a probe, where the probe hybridizes to a region of interest within a library member. The region of interest is separate from the adapter region and contains the genomic material of interest. The probe contains a ligand that allows the probe to be captured later. In some embodiments, the ligand preferably includes a biotin group.

The library is contacted with a probe in the presence of a hybridization buffer. In some embodiments, the hybridization buffer can include a crowding agent, as described in this disclosure. In some embodiments, hybridization buffers can include blockers as described in this disclosure.

In some embodiments, the method of hybrid capture includes a thermal denaturation step. For example, a hybridization mixture containing library members, blockers, and probes in a hybridization buffer can be heated to at least 90°C, at least 92°C, or at least 95°C. In some embodiments, the hybridization mixture can be heated up to 92°C, up to 95°C, up to 97°C or up to 100°C. In some embodiments, the hybridization mixture preferably further comprises Cot-1 DNA. In some embodiments, the hybridization buffer may include a blocker prior to addition to the hybridization mixture. In some embodiments, blockers can be added to the hybridization mixture independently of the hybridization buffer.

In some embodiments, the DNA concentration in the hybridization mixture (e.g., final hybridization reaction) is at least 0.1 ng/μL, at least 0.3 ng/μL, at least 0.4 ng/μL, at least 0. .5 ng/μL, at least 1 ng/μL, at least 2 ng/μL, at least 4 ng/μL, at least 6 ng/μL, at least 8 ng/μL, at least 10 ng/μL, at least 12 ng/μL, at least 15 ng/μL, at least 20 ng/μL, It can be at least 50 ng/μL, at least 75 ng/μL, at least 100 ng/μL, at least 120 ng/μL, at least 150 ng/μL, at least 200 ng/μL. In some embodiments, the DNA concentration in the hybridization mixture (e.g., final hybridization reaction) is up to 0.3 ng/μL, up to 0.4 ng/μL, up to 0.5 ng/μL, up to 1 ng /μL, maximum 2ng/μL, maximum 4ng/μL, maximum 6ng/μL, maximum 8ng/μL, maximum 10ng/μL, maximum 12ng/μL, maximum 15ng/μL, maximum 20ng/μL, maximum 50ng/μL, maximum 75ng /μL, up to 100ng/μL, up to 120ng/μL, up to 150ng/μL, up to 200ng/μL or up to 500ng/μL. For example, in some embodiments, the DNA concentration in the hybridization mixture (eg, final hybridization reaction) can range from 0.1 ng/μL to 120 ng/μL.

In some embodiments, the method includes a volume of the hybridization mixture of up to 150 μL, up to 140 μL, up to 130 μL, up to 120 μL, up to 110 μL, up to 100 μL, up to 90 μL, up to 80 μL, up to 70 μL, up to 60 μL, or up to 50 μL. may include use. In some embodiments, the method can include using a volume of the hybridization mixture of at least 10 μL, at least 20 μL, at least 30 μL, at least 40 μL, at least 50 μL, at least 60 μL, or at least 70 μL.

In some embodiments, the hybridization mixture is at least 1°C/min, at least 1.5°C/min, at least 2°C/min, at least 2.5°C/min, at least 3°C/min, or at least 5°C/min. It can be heated at a rate of 1 minute. In some embodiments, the hybridization mixture is heated up to 1.5°C/min, up to 2°C/min, up to 2.5°C/min, up to 3°C/min, up to 5°C/min or up to 10°C/min. It can be heated at a rate of 1 minute. In some embodiments, the hybridization mixture can be maintained at the temperature for thermal denaturation for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 20 minutes. In some embodiments, the hybridization mixture can be maintained at the temperature for thermal denaturation for up to 2 minutes, up to 5 minutes, up to 10 minutes, up to 20 minutes or longer.

After the heat denaturation step, the method of hybrid capture includes cooling the hybridization mixture. When the hybridization mixture is cooled from the thermal denaturation step temperature to the hybridization temperature, probe:target hybrids (ie, probe:library member hybrids) are formed.

In some embodiments, the hybridization temperature is at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C. ℃, at least 64 ℃, at least 65 ℃ or at least 70 ℃. In some embodiments, the hybridization temperature can be up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C or up to 70°C. . In some embodiments, hybridization can include reducing the temperature from a denaturing temperature to a hybridization temperature. In some embodiments, the temperature gradient is at least -5°C/min, at least -2°C/min, at least -1°C/min, at least -0.5°C/min, or at least -0.2°C/min. may include the speed of

In some embodiments, the method includes maintaining the library in contact with the probe and/or maintaining the hybridization mixture at the hybridization temperature for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours. , up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 90 minutes, up to 1 hour or up to 30 minutes. In some embodiments, the method includes maintaining the library in contact with the probe and/or heating the hybridization mixture at the hybridization temperature for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes. , at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours or at least 24 hours. In some embodiments, including where the T _m of the blocker is higher than the T _m of the adapter, the blocker:adapter hybrid forms before the adapter:adapter hybrid forms, thereby creating a "daisy chain" of adapter:adapter hybrids. formation or chain formation is prevented.
capture

In some embodiments, the method of hybrid capture includes capturing probes hybridized to members of the library using a capture means. The probe may be captured by any suitable means. For example, when the probe includes a biotin group, the probe can be captured using streptavidin beads, such as streptavidin-coated magnetic beads. In some embodiments, the probe may include FITC and the capture means may include anti-FITC. In some embodiments, the probe may include a hapten and the capture means may include a molecule (eg, an antibody) that binds to the hapten. Other capture means can also be used, such as isotachophoresis, size exclusion chromatography, liquid chromatography, magnetic means, and the like.

In some embodiments, such as when the capture means comprises streptavidin-coated beads, the method forms a capture mixture comprising a probe (e.g., a probe:target hybrid, etc.) and a capture means. may include steps.

In some embodiments, the method may include maintaining the capture mixture at a capture temperature. In some embodiments, the capture temperature is at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C. ℃, at least 64 ℃, at least 65 ℃ or at least 70 ℃. In some embodiments, the capture temperature can be up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C or up to 70°C. . In some embodiments, the capture mixture may be maintained at the capture temperature for at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes. In some embodiments, the capture mixture can be maintained at the capture temperature for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 30 minutes, up to 45 minutes or longer. In some embodiments, and/or shaking mixing may be added every 5 or 10 minutes.

In some embodiments, the method may further include washing the captured probe (the captured probe may include, for example, a probe:target hybrid and/or a captured library member). In some embodiments, captured probes can be washed at least once, at least two times, at least three times, or more. In some embodiments, captured probes can be washed up to 1 time, up to 2 times, up to 3 times, or up to 5 times.

In some embodiments, washing the captured library members can include heating the captured probes in a wash buffer to a wash temperature. In some embodiments, the wash buffer may include a detergent. Any suitable buffer may be used, including, for example, Tris-HCl; piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); piperazine-N,N'-bis(3-ethanesulfonic acid); -propanesulfonic acid) (PIPPS); 2-(N-morpholino)ethanesulfonic acid (MES); (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and the like. Any suitable surfactant may be used, including, for example, TWEEN® 20, TWEEN® 80, sodium dodecyl sulfate (SDS), and the like. In some embodiments, the wash buffer may include a compound aimed at reducing the formation of secondary structures in GC-rich regions, such as betaine. In some embodiments, the wash buffer can include an iron chelator, including, for example, deferoxamine mesylate, EGTA, EDTA, and the like.

The cleaning temperature is at least 20°C, at least 23°C, at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C. °C, at least 59 °C, at least 60 °C, at least 61 °C, at least 62 °C, at least 63 °C, at least 64 °C, at least 65 °C or at least 70 °C. In some embodiments, the wash temperature is up to 30°C, up to 35°C, up to 40°C, up to 45°C, up to 50°C, up to 55°C, up to 56°C, up to 60°C, up to 61°C, up to 62°C , up to 63°C, up to 64°C, up to 65°C or up to 70°C.

In some embodiments, the method may further include eluting captured library members from the capture means and/or probe. For example, captured library members can be eluted from streptavidin beads and/or biotinylated probes. In embodiments where the streptavidin beads include streptavidin magnetic beads, elution may include the use of a magnet. In some embodiments, elution can include the use of strong bases, including, for example, NaOH, KOH, Ca(OH) ₂ , and the like.

In some embodiments, a solid support may be used as a capture means, including, for example, a solid support comprising streptavidin. In some embodiments, the capture means may be washed under conditions of successively increasing stringency at a temperature below the estimated T _m value of the probe:target and/or at a temperature above the T _m value of the adapter. .

In some embodiments, the captured library members may be eluted from the ligand (e.g., from a biotinylated probe) or from the capture means (e.g., from streptavidin beads) and placed on a flow cell. can be loaded directly into

In some embodiments, at least 60 femtomoles, at least 80 femtomoles, at least 90 femtomoles, at least 100 femtomoles, or at least 150 femtomoles of DNA may be eluted. In some embodiments, up to 150 femtomoles, up to 500 femtomoles, up to 1 pmoles, up to 2 pmoles, or up to 3 pmoles of DNA can be eluted. For example, in some embodiments, 100 femtomoles to 2 pmoles of DNA may be eluted.

In some embodiments, eluted library members can be loaded onto a flow cell in a volume of less than 100 μL, less than 90 μL, less than 80 μL, less than 70 μL, or less than 60 μL. In some embodiments, captured library members can be loaded onto the flow cell in a volume of approximately 55 μL.

In some embodiments, after elution, the eluent can have a DNA concentration of at least 1.1 picomolar (pM), at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM. In some embodiments, the eluent can have a DNA concentration of up to 100 pM, up to 200 pM, up to 250 pM, or up to 300 pM. For example, in some embodiments, the eluent can have a DNA concentration within the range of 1.3 pM to 250 pM. In some embodiments, the eluent can be loaded onto the flow cell without further dilution and/or without changing the DNA concentration.
amplification

In another aspect, the disclosure describes a method that includes amplifying library members using PCR after hybrid capture but before sequencing the captured library members. Alternatively, the present disclosure provides methods that do not include subjecting the captured library members to amplification conditions after hybrid capture and before sequencing the library members (e.g., prior to sequencing the library members, A method that does not include the step of subjecting the captured library members to a PCR step to amplify the captured library members is described. Methods for sequencing captured library members typically include amplifying the library members (eg, using a PCR step) before sequencing said library members.

The amount of DNA captured during hybridization and hybrid capture may be too small to be directly quantified by fluorometric or analytical methods (eg, a bioanalyzer). Library amplification allows for quantification and quality control of the process.

Additionally, it is common to dilute samples amplified using PCR after hybrid capture and before sequencing. For example, after PCR, Illumina, Inc. Typical quantitation of NEXTERA Rapid Capture samples is performed in the mid-nanomolar range, whereas sequencing concentrations are in the low picomolar range. Thus, amplification of captured library members produces orders of magnitude more molecules than are needed for sequencing.

In some embodiments, the present disclosure describes methods of sequencing captured library members after hybrid capture. Such methods include loading captured and/or eluted library members directly onto a flow cell (e.g., U.S. Provisional Application No. 62/564,466 filed September 28, 2017). (e.g., using a direct flow cell loading jig) as described in .

In some embodiments, the method may further include sequencing the captured library members after hybrid capture.
Embodiments Exemplary Blocker Embodiments

1. A blocker comprising an oligonucleotide,
The blocker can bind to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The index area of the adapter and/or the area of the blocker that can be bound to the UMI are:
at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases; or A blocker comprising a universal base and at least one non-universal base.

2. The blocker according to blocker embodiment 1, wherein the region of the blocker capable of binding to the index region of the adapter or UMI contains the same number of thymine bases as the number of bases in the index region and/or UMI of the adapter.

3. The at least one non-universal base is located in a region of the blocker sequence corresponding to the index region and/or the UMI, and the non-universal base is located at the third or fifth position of the blocker sequence with respect to the 5' end of the index region. The blocker of embodiment 1, wherein the blocker is present at or both positions.

4. The blocker of Blocker Embodiment 1 or Blocker Embodiment 3, wherein the at least one non-universal base comprises a modified guanine.

5. 5. The blocker of any one of blocker embodiments 1, 3, or 4, wherein the at least one non-universal base comprises a random nucleotide.

6. Blocker embodiments 1, 3, 4 or 5, wherein the universal base comprises 2'-deoxyinosine, 2'-deoxynebularine, 3-nitropyrrole 2'-deoxynucleoside or 5-nitroindole 2'-deoxynucleoside. A blocker described in any one of the following.

7. The universal primer sequences are P5, P7, P5', P7', V2. A14. METS, V2. B15. METS, V2. A14. Complementary strand of METS and V2. B15. A blocker according to any one of the preceding blocker embodiments, comprising at least one complementary strand of METS.

8. A blocker as in any one of the preceding blocker embodiments, wherein said blocker comprises a modification that increases the T _m of said blocker compared to the same blocker without the modification.

9. DNA or RNA oligonucleotides modified such that the blocker captures low GC regions; bridge oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids. (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. , a blocker according to any one of the preceding blocker embodiments.

10. The blocker as in any one of the preceding blocker embodiments, wherein the blocker comprises a modified base in every base, at least every second base, at least every third base, at least every fourth base, or at least every fifth base.

11. A blocker according to any one of the preceding blocker embodiments, wherein said blocker comprises a 3' terminal group that prevents the availability of the blocker to serve as a primer for DNA synthesis.

12. The blocker of blocker embodiment 11, wherein the 3' end group comprises 3'-dC, 2',3'-ddC, inverted dT or 3'-spacer C3.

13. The blocker according to any one of the preceding blocker embodiments, wherein the blocker comprises the sequence of Table 2A, Table 2B, Table 2C, or Table 3.

14. A kit comprising a blocker according to any one of the preceding blocker embodiments.
Exemplary split blocker embodiment

1. A blocker comprising two unconnected oligonucleotides,
The blocker is capable of binding to at least a portion of an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The blocker, wherein the unconnected oligonucleotide contains bases that do not correspond to the index region and/or UMI of the adapter.

2. The blocker according to split blocker embodiment 1, wherein the unconnected oligonucleotide does not contain bases corresponding to the index region and/or UMI of the adapter.

3. The universal primer sequences are P5, P7, P5', P7', V2. A14. METS, V2. B15. METS, V2. A14. Complementary strand of METS and V2. B15. A blocker according to any one of the preceding split blocker embodiments, comprising at least one complementary strand of METS.

4. A blocker as in any one of the preceding split blocker embodiments, wherein the blocker includes a modification that increases the T _m of the blocker compared to the same blocker without the modification.

5. DNA or RNA oligonucleotides modified such that the blocker captures low GC regions; bridge oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids. (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. , a blocker as in any one of the preceding split blocker embodiments.

6. The blocker as in any one of the preceding split blocker embodiments, wherein the blocker comprises a modified base in every base, at least every second base, at least every third base, at least every fourth base, or at least every fifth base.

7. A blocker according to any one of the preceding split blocker embodiments, wherein said blocker comprises a 3' end group that prevents the availability of the blocker to serve as a primer for DNA synthesis.

8. The blocker of split blocker embodiment 11, wherein the 3' end group comprises 3'-dC, 2',3'-ddC, inverted dT or 3'-spacer C3.

9. The blocker according to any one of the preceding split blocker embodiments, wherein the blocker comprises the sequence of Table 2A, Table 2B, Table 2C, or Table 3.

10. The blocker of any of the split blocker embodiments, wherein the blocker comprises at least one of BN023, BN024, BN027 and BN028 of Table 2A.

11. A kit comprising a blocker according to any one of the preceding split blocker embodiments.
Exemplary hybridization buffer embodiments

1. Hybridization buffer containing crowding agent.

2. The hybridization buffer of Hybridization Buffer Embodiment 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine.

3. A hybridization buffer implementation as described above, wherein said crowding agent is included in said hybridization buffer in an amount of at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3% or at least 4%. Hybridization buffer according to any one of the forms.

4. The crowding agent is present in the hybridization buffer in an amount of at most 1%, at most 1.5%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 8% or at most 10%. A hybridization buffer according to any one of the preceding hybridization buffer embodiments, comprised in a hybridization buffer according to any one of the preceding hybridization buffer embodiments.

5. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

6. Hybridization buffer according to embodiment 5, wherein the hybridization buffer comprises Cot-1 DNA in an amount of at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL or at least 0.3 mg/mL. hybridization buffer.

7. Hybridization buffer embodiment 5 or 6, wherein the hybridization buffer comprises Cot-1 DNA in an amount of up to 0.1 mg/mL, up to 0.2 mg/mL, up to 0.3 mg/mL or up to 0.5 mg/mL. Hybridization buffer as described in .

8. Hybridization buffer embodiments 5-7, wherein the destabilizing agent constitutes at least 1% (v/v), at least 5% (v/v) or at least 10% (v/v) of the hybridization buffer. Hybridization buffer according to any one of.

9. Hybridization buffer embodiments 5-8, wherein said destabilizing agent constitutes up to 5% (v/v), up to 10% (v/v) or up to 15% (v/v) of said hybridization buffer. Hybridization buffer according to any one of.

10. Hybridization buffer according to any one of hybridization buffer embodiments 5-9, wherein the destabilizing agent comprises formamide.

11. The hybridization buffer is at least 0.1M, at least 0.2M, at least 0.3M, at least 0.4M, at least 0.5M, at least 0.6M, at least 0.7M, at least 0.8M, at least 0.9M. or a hybridization buffer according to any one of hybridization buffer embodiments 5-10, comprising at least 1M salt.

12. The hybridization buffer is maximum 0.2M, maximum 0.3M, maximum 0.4M, maximum 0.5M, maximum 0.6M, maximum 0.7M, maximum 0.8M, maximum 0.9M, maximum 1M, maximum Hybridization buffer according to any one of hybridization buffer embodiments 5-11, comprising 2M, up to 3M or up to 4M salt.

13. Hybridization buffer according to any one of hybridization buffer embodiments 5-11, wherein the salt comprises NaCl.

14. The hybridization buffer according to any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises phosphate.

15. The hybridization buffer of Hybridization Buffer Embodiment 14, wherein the hybridization buffer comprises at least 40 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM or at least 70 mM phosphate.

16. Hybridization buffer according to hybridization buffer embodiment 14 or (of) 15, wherein the hybridization buffer comprises at most 50 mM, at most 55 mM, at most 60 mM, at most 65 mM, at most 70 mM, at most 75 mM or at most 80 mM phosphate. .

17. Hybridization buffer according to any one of hybridization buffer embodiments 14-16, wherein the phosphate comprises KH ₂ PO ₄ -K ₂ HPO ₄ .

18. The hybridization buffer according to any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises a detergent.

19. the hybridization buffer is at least 0.001% (v/v), at least 0.01% (v/v), at least 0.05% (v/v), at least 0.1% (v/v); Hybridization Buffer The hybridization buffer of embodiment 18, comprising at least 1% (v/v) or at least 5% (v/v) surfactant.

20. The hybridization buffer may contain up to 0.01% (v/v), up to 0.05% (v/v), up to 0.1% (v/v), up to 1% (v/v), up to 5 % (v/v) or up to 10% (v/v) of detergent.

21. Hybridization buffer according to any one of hybridization buffer embodiments 18-20, wherein the surfactant comprises TWEEN® 20.

22. The hybridization buffer is
0.5% to 10% dextran sulfate,
0.05mg/mL to 0.5mg/mL human Cot-1 DNA,
1% to 15% (v/v) formamide,
40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ ,
0.1M to 4M NaCl, and 0.001% to 10% (v/v) Tween® 20
A hybridization buffer according to any one of the preceding hybridization buffer embodiments, comprising:

23. The hybridization buffer is
1.5% dextran sulfate,
0.2 mg/mL human Cot-1 DNA,
10% (v/v) formamide,
_{66.6mM KH2PO4} - _{K2HPO4 ,}
0.2mM enhanced adapter blocker (0.1mM for each adapter)
0.8M NaCl, and 0.04% (v/v) Tween® 20
A hybridization buffer according to any one of the preceding hybridization buffer embodiments, comprising:

24. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises a blocker.

25. Hybridization buffer of embodiment 24, wherein the blocker comprises a T _m elevated oligonucleotide.

26. 26. The hybridization buffer of hybridization buffer embodiment 24 or 25, wherein the blocker comprises multiple modifications that increase the _Tm .

27. Multiple modifications that increase the T _m of the blocker include: bridged oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA). a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5 modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. Hybridization buffer.

28. the hybridization buffer contains a blocker,
The blocker comprises an oligonucleotide;
The blocker can be attached to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The index region of the adapter and/or the region of the blocker that can bind to the UMI are:
at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases; or A hybridization buffer according to any one of the preceding hybridization buffer embodiments, comprising a universal base and at least one non-universal base.

29. the hybridization buffer contains a blocker,
the blocker comprises two unconnected oligonucleotides;
The blocker is capable of binding to at least a portion of an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
A hybridization buffer according to any one of the preceding hybridization buffer embodiments, wherein the unconnected oligonucleotide comprises a base that does not correspond to the index region of the adapter and/or the UMI of the adapter.

30. 30. The hybridization buffer of hybridization buffer embodiment 28 or 29, wherein the blocker comprises multiple modifications that increase the _Tm .

31. A kit comprising a hybridization buffer according to any one of the preceding hybridization buffer embodiments.
Exemplary Methods Using Hybridization Buffers (Buffer Method Embodiments)

1. A method comprising using a hybridization buffer containing a crowding agent for hybrid capture.

2. The method of buffer method embodiment 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine.

3. The crowding agent may be at least 0.5% (w/v), at least 1% (w/v), at least 1.5% (w/v), at least 2% (w/v), at least 3% ( or at least 4% (w/v) in said hybridization buffer.

4. The crowding agent may contain up to 1% (w/v), up to 1.5% (w/v), up to 2% (w/v), up to 3% (w/v), up to 4% (w/v). v), included in said hybridization buffer in an amount of up to 5% (w/v), up to 6% (w/v), up to 8% (w/v) or up to 10% (w/v); A method according to any one of the buffer method embodiments of.

5. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises at least one of human Cot-1 DNA, a destabilizing agent, and a salt.

6. Buffer method according to embodiment 5, wherein the hybridization buffer comprises human Cot-1 DNA in an amount of at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL or at least 0.3 mg/mL. the method of.

7. Buffer method embodiment 5 or 6, wherein the hybridization buffer comprises human Cot-1 DNA in an amount of up to 0.1 mg/mL, up to 0.2 mg/mL, up to 0.3 mg/mL or up to 0.5 mg/mL. The method described in.

8. Buffer method embodiments 5-7, wherein the destabilizing agent constitutes at least 1% (v/v), at least 5% (v/v) or at least 10% (v/v) of the hybridization buffer. Any one of the methods.

9. of buffer method embodiments 5-8, wherein said destabilizing agent constitutes at most 5% (v/v), at most 10% (v/v) or at most 15% (v/v) of said hybridization buffer. Any one of the methods.

10. The method of any one of buffer method embodiments 5-9, wherein the destabilizing agent comprises formamide.

11. The hybridization buffer is at least 0.1M, at least 0.2M, at least 0.3M, at least 0.4M, at least 0.5M, at least 0.6M, at least 0.7M, at least 0.8M, at least 0.9M. or at least 1M salt. The method of any one of buffer method embodiments 5-10.

12. The hybridization buffer is maximum 0.2M, maximum 0.3M, maximum 0.4M, maximum 0.5M, maximum 0.6M, maximum 0.7M, maximum 0.8M, maximum 0.9M, maximum 1M, maximum A method according to any one of buffer method embodiments 5-11, comprising 2M, up to 3M or up to 4M salt.

13. 13. The method of any one of buffer method embodiments 5-12, wherein the salt comprises NaCl.

14. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises phosphate.

15. 15. The method of buffer method embodiment 14, wherein the hybridization buffer comprises at least 40mM, at least 50mM, at least 55mM, at least 60mM, at least 65mM or at least 70mM phosphate.

16. 16. The method of buffer method embodiment 14 or 15, wherein the hybridization buffer comprises up to 50 mM, up to 55 mM, up to 60 mM, up to 65 mM, up to 70 mM, up to 75 mM or up to 80 mM phosphate.

17. 17. The method of any one of buffer method embodiments 14-16, wherein the phosphate comprises KH ₂ PO ₄ -K ₂ HPO ₄ .

18. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises a detergent.

19. the hybridization buffer is at least 0.001% (v/v), at least 0.01% (v/v), at least 0.05% (v/v), at least 0.1% (v/v); 18. The method of buffer method embodiment 17, comprising at least 1% (v/v) or at least 5% (v/v) surfactant.

20. The hybridization buffer may contain up to 0.01% (v/v), up to 0.05% (v/v), up to 0.1% (v/v), up to 1% (v/v), up to 5 % (v/v) or up to 10% (v/v) surfactant.

21. 21. The method of any one of buffer method embodiments 18-20, wherein the hybridization buffer comprises TWEEN® 20.

22. The hybridization buffer is
0.5% to 10% dextran sulfate,
0.05mg/mL to 0.5mg/mL Cot-1,
1% to 15% (v/v) formamide,
40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ ,
0.1M to 4M NaCl, and 0.001% to 10% (v/v) TWEEN® 20
A method according to any one of the preceding buffer method embodiments, comprising:

23. The hybridization buffer is
1.5% dextran sulfate,
0.2mg/mL Cot-1,
10% (v/v) formamide,
_{66.6mM KH2PO4} - _{K2HPO4 ,}
0.8M NaCl, and 0.04% (v/v) TWEEN® 20
A method according to any one of the preceding buffer method embodiments, comprising:

24. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises a blocker.

25. 25. The method of buffer method embodiment 24, wherein the blocker is present at a concentration of at least 0.001 mM, at least 0.005 mM, at least 0.01 mM, at least 0.05 mM, or at least 0.1 mM.

26. 26. The method of buffer method embodiment 24 or 25, wherein said blocker is present at a concentration of up to 0.01 mM, up to 0.05 mM, up to 0.1 mM, up to 0.2 mM or up to 0.5 mM.

27. Any of the preceding buffer method embodiments, wherein the method comprises forming the hybridization buffer from a first composition comprising human Cot-1 DNA and a blocker and a second composition comprising formamide and dextran sulfate. The method described in one.

28. The method as in any one of the preceding buffer method embodiments, wherein the method comprises contacting the library with the hybridization buffer, the hybridization buffer comprising a blocker.

29. 29. The method of buffer method embodiment 28, wherein the method comprises combining samples from at least two library preparations before contacting the library with the blocker in the presence of the hybridization buffer.

30. The method of buffer method embodiment 29, wherein at least 1 μL, at least 2 μL, at least 3 μL, at least 5 μL, at least 10 μL, at least 15 μL, or at least 20 μL of the sample from each library preparation is combined.

31. Buffer method embodiment 29 or 30, combining up to 2 μL, up to 3 μL, up to 5 μL, up to 10 μL, up to 15 μL, up to 20 μL, up to 25 μL, up to 30 μL, up to 40 μL, or up to 50 μL of samples from each library preparation. The method described in.

32. of buffer method embodiments 29-31, wherein at least 10 ng, at least 15 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 500 ng, at least 1,000 ng or at least 5,000 ng of DNA from each library preparation is combined. Any one of the methods.

33. Combine up to 15ng, up to 25ng, up to 50ng, up to 100ng, up to 200ng, up to 500ng, up to 1,000ng, up to 5,000ng, up to 10,000ng or up to 12,000ng of DNA from each library preparation in buffer 33. The method according to any one of method embodiments 29-32.

34. the DNA concentration of each sample from the combined library preparations is at least 0.1 ng/μL, at least 0.3 ng/μL, at least 0.4 ng/μL, at least 0.5 ng/μL, at least 1 ng/μL, at least 2 ng /μL, at least 4ng/μL, at least 6ng/μL, at least 8ng/μL, at least 10ng/μL, at least 12ng/μL, at least 20ng/μL, at least 50ng/μL, at least 60ng/μL, at least 100ng/μL or at least 200ng /μL.

35. The DNA concentration of each sample from the combined library preparations is up to 0.4 ng/μL, up to 0.5 ng/μL, up to 1 ng/μL, up to 2 ng/μL, up to 4 ng/μL, up to 6 ng/μL, up to 8ng/μL, maximum 10ng/μL, maximum 12ng/μL, maximum 15ng/μL, maximum 20ng/μL, maximum 50ng/μL, maximum 100ng/μL, maximum 120ng/μL, maximum 200ng/μL, maximum 300ng/μL or maximum The method of any one of buffer method embodiments 29-34, wherein the buffer is 400 ng/μL.

36. 36. The method of any one of buffer method embodiments 29-35, wherein the DNA concentration after combining the library preparations is in the range of 0.3 ng/μL to 400 ng/μL.

37. Buffer method embodiments 29-36, wherein the method further comprises contacting the library member with a probe, the probe hybridizing to a region of interest within the library member, and the probe comprising a ligand. Any one of the methods.

38. 38. The method of buffer method embodiment 37, wherein the ligand comprises a biotin group.

39. Any one of buffer method embodiments 29-38, wherein the method comprises forming a hybridization mixture comprising a library member, the hybridization buffer (wherein the hybridization buffer comprises a blocker), and a probe. The method described in.

40. 40. The method of buffered method embodiment 39, wherein the hybridization mixture further comprises human Cot-1 DNA.

42. The method comprises using a volume of the hybridization mixture of at most 150 μL, at most 140 μL, at most 130 μL, at most 120 μL, at most 110 μL, at most 100 μL, at most 90 μL, at most 80 μL, at most 70 μL, at most 60 μL, or at most 50 μL. A method according to method embodiment 39 or 40.

42. Any one of buffer method embodiments 39-41, wherein the method comprises using a volume of the hybridization mixture of at least 10 μL, at least 20 μL, at least 30 μL, at least 40 μL, at least 50 μL, at least 60 μL, or at least 70 μL. Method described.

43. The DNA concentration in the hybridization mixture is at least 0.1 ng/μL, at least 0.3 ng/μL, at least 0.4 ng/μL, at least 0.5 ng/μL, at least 1 ng/μL, at least 2 ng/μL, at least 4 ng/μL, at least 6 ng/μL, at least 8 ng/μL, at least 10 ng/μL, at least 12 ng/μL, at least 15 ng/μL, at least 20 ng/μL, at least 50 ng/μL, at least 75 ng/μL, at least 100 ng/μL, at least 43. The method of any one of buffer method embodiments 39-42, wherein the buffer is 120 ng/μL, at least 150 ng/μL, at least 200 ng/μL.

44. The DNA concentration in the hybridization mixture is a maximum of 0.3 ng/μL, a maximum of 0.4 ng/μL, a maximum of 0.5 ng/μL, a maximum of 1 ng/μL, a maximum of 2 ng/μL, a maximum of 4 ng/μL, a maximum of 6 ng/μL , maximum 8ng/μL, maximum 10ng/μL, maximum 12ng/μL, maximum 15ng/μL, maximum 20ng/μL, maximum 50ng/μL, maximum 75ng/μL, maximum 100ng/μL, maximum 120ng/μL, maximum 150ng/μL , up to 200 ng/μL, or up to 500 ng/μL.

45. The library may be exposed to the probe for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 90 minutes, up to 1 The method of any one of buffer method embodiments 37-44, wherein the buffer is contacted for an hour or up to 30 minutes.

46. The library has been with the probe for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours, or at least 24 hours. 46. The method of any one of buffer method embodiments 37-45, wherein the buffer is contacted for a period of time.

47. The method includes maintaining the hybridization mixture at a hybridization temperature, the hybridization temperature being at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C. 47. The method of any one of buffer method embodiments 39-46, wherein the buffer temperature is at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C or at least 70°C.

48. The method includes maintaining the hybridization mixture at a hybridization temperature, wherein the hybridization temperature is up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C. 48. The method of any one of buffer method embodiments 39-47, wherein the buffer temperature is at most 65°C or at most 70°C.

49. The method includes heating the hybridization mixture at the hybridization temperature for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 49. The method of any one of buffer method embodiments 39-48, comprising maintaining for 90 minutes, up to 1 hour or up to 30 minutes.

50. The method comprises heating the hybridization mixture at the hybridization temperature for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 50. The method of any one of buffer method embodiments 39-49, comprising maintaining for 20 hours or at least 24 hours.

51. 51. The method of any one of buffer method embodiments 37-50, wherein the method further comprises capturing the hybridized probe using a capture means.

52. 52. The method of buffered method embodiment 51, wherein capturing the probe comprises using streptavidin beads.

53. 53. The method of any one of buffer method embodiments 37-52, wherein said method comprises forming a capture mixture comprising said probe and capture means.

54. The method comprises capturing the capture mixture at a temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C. 54. The method of buffer method embodiment 53, comprising maintaining a capture temperature of at least 65°C or at least 70°C.

55. The method maintains the capture mixture at a capture temperature of up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C or up to 70°C. 55. The method of buffer method embodiment 53 or 54, comprising:

56. 56. The method of any one of buffer method embodiments 53-55, wherein the method comprises maintaining the capture mixture at the capture temperature for at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes.

57. Any of buffer method embodiments 53-56, wherein the method comprises maintaining the capture mixture at the capture temperature for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 30 minutes, or up to 45 minutes. The method described in one.

58. 58. The method of any one of buffer method embodiments 53-57, wherein the method includes washing the captured probe.

59. 59. The method of any one of buffer method embodiments 53-58, wherein the method further comprises eluting the captured library member from the capture means in an eluent.

60. 60. The method of any one of buffer method embodiments 53-59, wherein the method further comprises eluting the captured library member from the probe in an eluent.

61. 61. The method of buffer method embodiment 60, wherein at least 60 femtomoles, at least 80 femtomoles, at least 90 femtomoles, at least 100 femtomoles, or at least 150 femtomoles of DNA are eluted.

62. 62. The method of buffer method embodiment 60 or 61, wherein at most 150 femtomoles, at most 500 femtomoles, at most 1 pmol, at most 2 pmoles or at most 3 pmoles of DNA is eluted.

63. 63. The method of any one of buffered method embodiments 60-62, wherein the DNA concentration of the eluent is at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM.

64. 64. The method of any one of buffer method embodiments 60-63, wherein the DNA concentration of the eluate is at most 100 pM, at most 200 pM, at most 250 pM, or at most 300 pM.

65. 65. The method of any one of buffer method embodiments 60-64, wherein the DNA concentration of the eluate is within the range of 1.3 pM to 250 pM.

66. 66. The method of any one of buffer method embodiments 37-65, wherein said method further comprises sequencing said library member.

67. 67. The method of buffer method embodiment 66, wherein the method comprises amplifying the library member prior to sequencing the library member.

68. 67. The method of buffer method embodiment 66, wherein the method does not include amplifying the library member before sequencing the library member.

69. 69. The method of any one of buffer method embodiments 24-68, wherein the blocker comprises a T _m elevated oligonucleotide.

70. 70. The method of any of buffered method embodiments 24-69, wherein the blocker comprises multiple modifications that increase T _m .

71. The plurality of modifications that increase the T _m of the blocker include: bridged oligonucleotide; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA); ); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5 modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. Method.

72. the blocker comprises an oligonucleotide;
the blocker is capable of binding to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The index region of the adapter and/or the region of the blocker that can bind to the UMI are:
at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases; or 72. The method of any of buffer method embodiments 24-71, comprising a universal base and at least one non-universal base.

73. the blocker comprises two unconnected oligonucleotides;
the blocker is capable of binding to at least a portion of an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
73. The method of any of buffer method embodiments 24-72, wherein the unconnected oligonucleotide comprises a base that does not correspond to the index region of the adapter and/or the UMI of the adapter.
Exemplary Method of Hybrid Capture Without PCR (Capture Method Embodiment)

1. contacting the library with a blocker in the presence of a hybridization buffer;
and contacting the library with a probe, the probe hybridizing to a region of interest within a library member;
The method does not include the step of amplifying the library member using PCR before sequencing the library member.

2. 2. The method of capture method embodiment 1, wherein the probe further comprises a ligand, and the library member is eluted from the ligand prior to sequencing the library member.

3. The method of capture method embodiment 2, wherein the ligand comprises biotin.

4. 4. The method of any of capture method embodiments 2 or 3, wherein the method comprises loading the library member onto a flow cell after elution from the ligand.

5. The method according to any one of capture method embodiments 1 to 4, wherein the method further includes the step of capturing the hybridized probe using a capture means.

6. 6. The method of capture method embodiment 5, wherein the capture means comprises streptavidin.

7. 7. The method of any of capture method embodiments 5 or 6, wherein the method comprises loading the library member onto a flow cell after elution from the capture means.

8. Capture method embodiment 4 or 7, wherein the method comprises loading the library member onto a flow cell in a volume of less than 100 μL, a volume of less than 90 μL, a volume of less than 80 μL, a volume of less than 70 μL, or a volume of less than 60 μL. The method described in.

9. Capture method according to embodiments 4 or 7, wherein the method comprises loading the library member onto a flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM. the method of.

10. 10. The method of capture method embodiment 4, 7 or 9, wherein the method comprises loading the library member onto a flow cell at a concentration of up to 100 pM, up to 200 pM, up to 250 pM or up to 300 pM.

11. 11. The method of any one of capture method embodiments 4 or 7-10, wherein the method comprises loading the library member onto a flow cell using a direct flow cell loading jig.

12. The method of any one of the preceding capture method embodiments, wherein the hybridization buffer comprises a crowding agent.

13. The hybridization buffer is
0.5% to 10% dextran sulfate,
0.05mg/mL to 0.5mg/mL human Cot-1 DNA,
1% to 15% (v/v) formamide,
40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ ,
0.1M to 4M NaCl, and 0.001% to 10% (v/v) Tween® 20
13. The method of capture method embodiment 12, comprising:

14. The method of any one of the preceding capture method embodiments, wherein the blocker comprises a T _m elevated oligonucleotide.

15. The method of any one of the preceding capture method embodiments, wherein the blocker comprises multiple modifications that increase _Tm .

16. The plurality of modifications that increase the T _m of the blocker include: bridged oligonucleotide; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA); ); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5 modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. Method.

17. the blocker comprises an oligonucleotide;
the blocker is capable of binding to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The index region of the adapter and/or the region of the blocker capable of binding to the UMI,
at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases; or A method according to any one of the preceding capture method embodiments, comprising a universal base and at least one non-universal base.

18. the blocker comprises two unconnected oligonucleotides;
the blocker is capable of binding to at least a portion of an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI);
The method of any one of the preceding capture method embodiments, wherein the unattached oligonucleotide comprises a base that does not correspond to the index region of the adapter and/or the UMI of the adapter.

The invention is illustrated by the following examples. It is understood that specific examples, materials, amounts and procedures are to be broadly construed in accordance with the scope and spirit of the invention set forth herein.
Embodiment

The following numbered items provide embodiments as described herein, but the embodiments described herein are not limiting.

Item 1. A hybridization buffer comprising a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

Item 2. The hybridization buffer according to item 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine.

Item 3. The hybridization buffer includes 0.5% to 10% dextran sulfate, 0.05mg/mL to 0.5mg/mL Cot-1, 1% to 15% (v/v) formamide, 40mM to 80mM KH ₂ PO ₄ - Hybridization buffer according to any one of the preceding items, comprising K ₂ HPO ₄ , 0.1 M to 4 M NaCl, and 0.001% to 10% (v/v) TWEEN® 20.

Item 4. Hybridization buffer according to any one of the preceding items, wherein said blocker comprises multiple modifications that increase the _Tm .

Item 5. A method comprising using a hybridization buffer according to any one of the preceding items.

Item 6. 6. The method of item 5, wherein the method comprises forming a hybridization mixture comprising a library member, the hybridization buffer, human Cot-1 DNA, a blocker, and a probe.

Item 7. Item 6, wherein the hybridization mixture comprises samples from at least two library preparations, combining at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng or at least 500 ng of DNA from each library preparation. The method described in.

Item 8. The method according to item 6 or item 7, wherein the DNA concentration of the hybridization mixture is within the range of 0.1 ng/μL to 120 ng/μL.

Item 9. the method comprises contacting the library member with the probe, the probe hybridizing to a region of interest within the library member, the probe comprising a ligand; using a capture means, and eluting the captured library member from the capture means into an eluent, wherein the eluent has a DNA concentration of 1.3 pM to 250 pM. The method according to any one of items 6 to 8, comprising a step within the scope of.

Item 10. Any of items 6-9, wherein the method further comprises sequencing the library member, and wherein the method does not include amplifying the library member before sequencing the library member. The method described in one.

Item 11. 11. The method of item 10, wherein the method comprises loading the library member onto a flow cell using a direct flow cell loading jig.

Item 12. A blocker comprising an oligonucleotide, said blocker capable of binding to an adapter, said adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), said adapter comprising: The index region of the blocker and/or the region of the blocker that can bind to the UMI includes at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 1 thymine base, at least 9 thymine bases or at least 10 thymine bases; or a universal base and at least one non-universal base.

Item 13. A blocker comprising two unconnected oligonucleotides, said blocker capable of binding at least a portion of an adapter, said adapter comprising a universal primer sequence and an index region or unique molecular identifier (UMI). and wherein the unconnected oligonucleotide contains bases that do not correspond to the index region and/or UMI of the adapter.

Item 14. The universal primer sequences are P5, P7, P5', P7', V2. A14. METS, V2. B15. METS, V2. A14. Complementary strand of METS and V2. B15. The blocker according to item 12 or 13, comprising at least one complementary strand of METS.

Item 15. 15. A blocker according to any one of items 12 to 14, wherein the blocker comprises a modification that increases the T _m of the blocker compared to the same blocker without the modification.

Item 16. DNA or RNA oligonucleotides modified such that the blocker captures low GC regions; bridge oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids. (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. , the blocker according to any one of items 12 to 15.

Item 17. contacting a library with a blocker in the presence of a hybridization buffer; and contacting the library with a probe, the probe hybridizing to a region of interest within a library member; The method does not include the step of amplifying the library member using PCR before sequencing the library member.

Item 18. The method comprises loading the library member onto a flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM and at most 100 pM, at most 200 pM, at most 250 pM or at most 300 pM. The method according to item 17, comprising the step of:

Item 19. 19. The method of item 17 or 18, wherein the blocker comprises multiple modifications that increase _Tm .

Item 20. 20. The method according to any one of items 17-19, wherein the hybridization buffer comprises a crowding agent.

Item 21. The hybridization buffer includes 0.5% to 10% dextran sulfate, 0.05mg/mL to 0.5mg/mL human Cot-1 DNA, 1% to 15% (v/v) formamide, and 40mM to 80mM KH ₂ PO. 20. The method of any one of items 17-20, comprising ₄ -K ₂ HPO ₄ , 0.1 M to 4 M NaCl, and 0.001% to 10% (v/v) Tween 20.

Item 22. 22. The method of any one of items 17-21, wherein the method comprises loading the library member onto a flow cell using a direct flow cell loading jig.

Item 23. A kit comprising the hybridization buffer according to any one of items 1 to 4.

Item 24. A kit comprising the blocker according to any one of items 12 to 16.

Example 1 - Hybridization and Capture Hybridization Non-enriched hybridization buffer contains the following components/final concentrations in a 100 μL hybridization reaction: human Cot-1 0.2 mg/ml, formamide 10% (v /v), KH ₂ PO ₄ -K ₂ HPO ₄ 66.6 mM, NaCl 0.8 M, TWEEN® 20 0.04% (v/v).

The enhanced hybridization buffer contains the following components/final concentrations in a 100 μL hybridization reaction: dextran sulfate 1.5% (w/v), human Cot-1 0.2 mg/ml, formamide 10% ( v/v), KH ₂ PO ₄ -K ₂ HPO ₄ 66.6 mM, NaCl 0.8 M, TWEEN® 20 0.04% (v/v).

In each case, the 100 μL final reaction contains probes (at least 500 probes and up to 500,000 probes) at a concentration of 125 pM per probe (total probe concentration of at least 30 nM), and up to 30 μL (50 ng to 6 μg) of DNA sample. Blockers, if included, are present at 0.2mM.

Hybridization reactions are performed in a thermocycler or HYBEX system using precise temperature control. After 5 minutes of denaturation at 95°C, hybridization reactions are incubated at 58°C or 62°C for 90 minutes (total hybridization time approximately 100 minutes) or up to 24 hours. Depending on the probe design, incubation at 58°C or 62°C can be performed starting from 95°C to 58°C or 62°C, followed by a ramp of 2°C per minute (another 20 minutes). For the experiments described inside, incubate the samples at 62°C).
capture

Using 250 μL of streptavidin magnetic beads SMB (Streptavidin Magnetic Beads, Illumina, Inc., San Diego, CA) for each 100 μL of hybridization reaction, the hybridization incubation temperature (e.g., 62° C. If incubated for 90 minutes, capture the targeted DNA for 15 minutes at 62°C. The targeting DNA was then washed using wash buffer EEW (Enhanced Enrichment Wash Buffer, Illumina, Inc., San Diego, CA (Tris-HCL, deferoxamine mesylate (DFO), betaine and TWEEN® 20)). and wash at the same temperature (eg, 62°C) for 5 minutes a total of 4 times. Targeted DNA was eluted using fresh denaturing solution (0.1% (v/v) TWEEN® 20 (EE1) and 2N NaOH (HP3)) and ET2 (Elution Target Buffer 2, Illumina, Inc., San Diego, CA (Tris base, Tris acetate)), primer cocktail (PPC; sequencing adapter primer set containing 5 μM P5 and P7 primers, Illumina, Inc.) and Enhanced PCR Mix ( EPM) (Illumina, Inc., San Diego, CA) and cleanup using 0.9x Solid Phase Reversible Immobilization (SPRI) beads.
Example 2 - Blocker containing thymine

This example describes a blocker that includes thymine in the region of the blocker that corresponds to the index and/or UMI (see Figure 3D).

Hybridization and capture are performed as described in Example 1 using the blockers in Table 3, enhanced hybridization buffer and 0.2mM blocker. The resulting captured DNA is sequenced and Padded Read Enrichment is calculated using Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, Calif.).

Blockers containing a stretch of T in the position of the index sequence contain at least 8 or 10 modified nucleotides in regions of the blocker that do not correspond to the index to increase on-target DNA capture (as measured by Padded Read Enrichment). Requires.

Oligos containing sequences complementary to the index region required fewer modified nucleotides (8 and 10 nucleotides tested) and showed higher Padded Read Enrichment. The results are shown in Figure 4. If the blockers contain the same number of modifications (8 or 10 nucleotides), then the blockers containing a complementary sequence in the index region (last bar) will be the same as the blockers containing a stretch of T in the index region (in Figure 4 " 10 modifications and a higher Padded Read Enrichment than (labeled (T)8).
Example 3 - Blockers containing universal bases and modified bases

Hybridization and capture are performed as described in Example 1 using 0.2 mM of the blockers in Table 4 in enhanced hybridization buffer. As shown in Table 4, the region of the blocker corresponding to the index region contains a universal base (deoxyinosine) and an LNA modified guanine or random nucleotide.

While not wishing to be bound by theory, it is believed that modified G or random nucleotides are placed at every second base of the blocker sequence corresponding to the index region (e.g., as shown in one embodiment in FIG. or the second and/or fourth position of the blocker sequence corresponding to the index region). It is believed that blocker affinity is improved.
Example 4 - Split Blocker

This example shows that a split LNA blocker works well when using a library containing an 8 nucleotide index.

Hybridization and capture are performed as described in Example 1 using the blockers in Table 5 and using enhanced hybridization buffer. As shown in Table 5, the blockers TiLNAS1 (BN025), TiLNAS2 (BN026), P5LNA (BN023) and P7LNA (BN024) do not contain a region corresponding to the index region. P5LNA (BN023) and P7LNA (BN024) each contain eight LNA modified bases. TiLNAS1 (BN025) and TiLNAS2 (BN026) each contain 10 LNA modified bases. TiLNA17 (BN027) and TiLNA18 (BN028) contain a universal base (deoxyinosine) in the region of the blocker corresponding to the index region, but the regions of the blocker corresponding to other regions of the adapter are shortened.

The resulting captured DNA is sequenced and Padded Read Enrichment is calculated using Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, Calif.). Blocker diagrams are shown in FIGS. 6A and 3E-3I, and the results are shown in FIG. 6B. Sample "Split 4pc" contains TiLNAS1 (BN025), TiLNAS2 (BN026), P5LNA (BN023) and P7LNA (BN024) from Table 5; sample "Inner" contains TiLNAS1 (BN025) and TiLNAS2 (BN026) from Table 5. ); Sample “Outer” contains P5LNA (BN023) and P7LNA (BN024) in Table 5; Sample Short8nt contains TiLNA17 (BN027) and TiLNA18 (BN028) in Table 5. Two two-piece blockers (“split blockers”) containing BN023, BN024, BN025 and BN026 were tested by XGEN Blocking Oligos (Integrated DNA Technologies, Coralv) as measured by Padded Read Enrichment. ille, IA), and the adapter's Experiments using blockers that block only part of the disease are not likely to work as well.
Example 5 - Hybridization buffer

This example shows a comparison of on-target capture using non-enhanced and enhanced hybridization buffers.

Hybridization and capture are performed as described in Example 1 using XGEN Blocking Oligos (Integrated DNA Technologies, Coralville, IA) and enhanced hybridization buffer with dextran sulfate concentrations shown in Figure 7A. . The resulting captured DNA is sequenced and Padded Read Enrichment is calculated using Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, Calif.). The results are shown in Figure 7A. Inclusion of dextran sulfate allows rapid hybridization (60-90 minutes) in high volumes (100 μL) of hybridization buffer.

Enrichment using a 100 μL hybridization buffer volume was performed using four different probe panels: Coding Exome Oligos (CEX) (Illumina, Inc., San Diego, CA), IDT Exome Research Panel (IDT Exome) (Integrate d DNA Technologies, Coralville, IA), TRUSIGHT Cancer Panel (Cancer) (Illumina, Inc., San Diego, CA) and TRUSIGHT One Sequencing Panel (TSO) (Illumina, Inc.). , San Diego, CA). Hybridization is tested in three hybridization buffers: the IDT buffer of the XGEN LOCKDOWN kit (Integrated DNA Technologies, Coralville, IA), and the hybridization buffer with and without dextran sulfate as described in Example 1. The results are shown in Figure 7B. The enhanced hybridization buffer provides similar or higher on-target results compared to the IDT buffer for all four probe panels (ranging from 4,000 to 450,000 probes).

Two key enrichment metrics (Padded Read Enrichment and Coverage Uniformity) for four panels (CEX, IDT Exome, Cancer and TSO) at various hybridization incubation times, with and without an additional temperature gradient step. using Illumina enhanced hybridization buffer containing IDT XGEN blocker. The results are shown in Figure 7C. The black dotted line in the left panel shows the results of hybridization in buffer without dextran sulfate.

Three runs of 12-plex library input are pooled to a total volume of 30 μL and enriched using Illumina enhanced hybridization buffer containing IDT XGEN blocker. Samples were transferred to Illumina, Inc. on various sequencing machines (NOVASEQ, NEXTSEQ and ISEQ). No sample concentration step is required. The results are shown in Figures 7D-7F. The results show that when using an enriched hybridization buffer containing dextran sulfate, up to 30 μL of sample can be accommodated in one hybridization reaction (e.g., in a single 15 μL sample or in each 2.5 μL sample). 12-plex run). Enrich up to 12 samples in one reaction using enhanced hybridization buffers without using speed vacuums, centrifugation columns or SPRI beads to further reduce pool volumes. be able to.
Example 6 - Improved Adapter + Enhanced Hybridization Buffer

The enrichment performance of the IDT exome panel (Cat #1056114, Integrated DNA Technologies, Coralville, IA) was determined by (1) XGEN LOCKDOWN kit (Integrated DNA Technologies, Coralville, IA). , IA) IDT buffer (17 μL); (2) small ( (11 μL) non-enhanced hybridization buffer (TiE0) containing non-enhanced adapter blockers (NEXTERA Blocker1 i5 (BN001) and NEXTERA Blocker2 i7 (BN002) in Table 2) in a small (11 μL) reaction volume; (4) non-enhanced hybridization buffer (TiE3) containing an enhanced adapter blocker (XGEN blocker); (4) non-enhanced hybridization buffer (TiE4) containing an enhanced adapter blocker (XGEN blocker) in a large (100 μL) reaction volume; and (5) ) Test using enhanced hybridization buffer (TiE9) containing enhanced adapter blocker (XGEN blocker) in a large (100 μL) reaction volume. The results are shown in Figure 8A and demonstrate that enrichment performance is improved by using modified blockers, increased wash temperatures, and/or a crowding agent (dextran sulfate) in the hybridization buffer. .

Enrichment performance (measured using Padded Read Enrichment) was determined in nine probe panels using an IDT enrichment assay (Catalog No. 1080584, Integrated DNA Technologies, Coralville, IA) and enhanced hybridization buffer. In an Illumina enrichment assay using High Padded Read Enrichment is achieved in 100 μL reaction volumes containing various probe panels (500-450,000 probes). Probe panels include (1) XGEN Exome Research Panel (IDT Exome) (Catalog No. 1056114, Integrated DNA Technologies, Coralville, IA); (2) Twist Human C ore Exome (Twist Bioscience, Inc., Catalog No. 100254) (3) Coding Exome Oligos (CEX) (Catalog No. 15034575, Illumina, Inc., San Diego, CA); (4) TruSight One (Illumina Inc, San Diego, CA, Catalog) Log No. FC-141-1007; Probe Catalog No. 15046658); (5) TruSight One Expanded (Illumina, Inc. San Diego, Catalog No. FC-141-2007; Probe Catalog No. 20015656 ); (6) TruSight Cancer (Illumina, Inc., San Diego, CA, Catalog No. FC-121-0202; Probe Catalog No. 15035642); (7) TruSight Cardio (Illumina, Inc., San Diego, CA, Catalog No. FC-141-1010; P Robe Catalog No. 15069654); (8) TruSight RNA Fusion Panel (Illumina Inc, San Diego, CA, Catalog No. 20000906); (9) Oncology DNA Probes Master Pool (OPD1) (Illumin a, Inc., Catalog No. OP-101-1004; Probe Catalog No. 20001565). The results are shown in Figure 8B. The white bar (column 1) shows the performance of the IDT exome panel in the IDT lockdown enrichment workflow using Nextera Rapid Capture library preparations (Illumina, Inc., San Diego, Calif.). Gray bars indicate the performance of the panel when using the Illumina Enrichment Assay with enhanced hybridization buffer and IDT xGEN adapter blocker. The black horizontal lines indicate the results obtained using the Twist enrichment assay (Twist Bioscience, Inc.) (column 3), Nextera Rapid Capture library preparation (columns 4-8), or the TruSight Tumor 170 workflow (column 10) with the OPD1 panel. , shows the comparative results obtained in the corresponding panels. The performance of each panel in the Illumina Enrichment Assay with enhanced hybridization buffer is evaluated in at least three independent experiments; error bars indicate standard deviation.

A single hybridization enrichment protocol using enhanced hybridization buffer and XGEN blocker for a panel of 12 genes and 535 probes to enrich libraries generated from Horizon Discover HD701 quantitative multiplex (QM) DNA standards. to detect somatic variants. The data shown in Figure 8C shows expected variant frequency (x-axis) versus called variant frequency (y-axis). Each panel in Figure 8C shows various concentrations of high affinity adapter blocker (IDT (none). Each point represents a somatic variant call colored by read depth. The red dashed line represents the perfect correlation between known and called variant frequencies. The blue and gray shading represent the best-fit line and 95% confidence interval, respectively, for the called variant frequency when compared to the known variant frequency. The expected value of the variant frequency ( x-axis) correlates with known (called) variant frequencies (y-axis) for IDT XGEN adapter blockers (0.1×(0.02mM), 0.5×(0.1mM)) but negative control (no high affinity adapter blocker)).
Example 7A - PCR-free hybrid capture using TRUSEQ adapter ligated library

This example shows hybrid capture without PCR.

Hybridization is performed using XGEN LOCKDOWN reagent (Integrated DNA Technologies, Coralville, IA) according to package instructions with the following modifications. Targeting DNA is generated using the TRUSEQ adapter ligation approach and captured using 100 μL of DYNABEADS M-270 Streptavidin (Integrated DNA Technologies, Coralville, IA). This hybridization is incubated for only 30 minutes instead of the recommended 4 hours. Then follow the following protocol:
1. The beads are incubated with 10 μL of 0.1 N NaOH to denature the captured library from the probe and release it into solution.
2. Remove the captured library solution from the beads (using a magnet) and add 10 μL of 200 mM Tris. Neutralize with HCl pH 7.0.
3. (If necessary) Add up to 7.5 μL of resuspension buffer (RSB) (Illumina, Inc., San Diego, CA) to the sample with denatured PhiX Control v3 (Illumina, Inc., San Diego, CA). Add.
4. Add 27.5 uL of 2xHT1 (Hybridization Buffer, Illumina, Inc., San Diego, CA).

The resulting sample (in 55 μL of 1×HT1 buffer) was transferred using a pipette or flow cell loading jig, e.g., as described in U.S. Provisional Application No. 62/564,466, filed September 28, 2017. and load directly onto the flow cell. The results are shown in Table 6A. Controls (column 1) are performed using the same conditions as the no-PCR method, except that capture is followed by PCR. The first attempt to perform PCR-free enrichment is shown in column 2, indicating that there were too few reads to reach an acceptable level of coverage for the exome. While the goal was to exceed approximately 50×, only an average target coverage of approximately 18× was achieved. For the second attempt (column 3), using a direct flow cell loading jig and increasing the amount of DNA input for hybridization increased the sequencing rate of the PCR-free enrichment method by approximately 40×. This shows an improvement of about 140x, which is much higher than the target. Downsampling the no-PCR data to 70 million reads (column 4) yields a metric similar to (or better than) the control.
Example 7B - PCR-free hybrid capture using bead-based NEXTERA libraries improves exome quality with short hybridization times

Control experiments are performed using similar conditions to Example 7A, but with libraries generated using a bead-based tagmentation library preparation method and corresponding blocking oligonucleotides. Both the duration of hybridization and PCR amplification are variables in this experiment. Recommended 4-hour hybridization reaction protocol (7.5 hours total, see IDT , used as a control (a schematic of this workflow is shown in Figure 9B) and compared to two conditions using only 30 minutes of hybridization (with and without post-capture PCR) and direct loading into the flow cell (Figure 9C ). By shortening the duration of hybridization and eliminating PCR, the enrichment protocol can be performed in only about 2 hours. A schematic diagram of this workflow, which reduces hybridization time and does not involve PCR, is shown in Figure 9D.

The results are shown in Table 6B. Downsample all data to approximately 23 million clusters. In the first column (control), the data shows good quality exome, but with a long workflow of about 7.5 hours. Reducing hybridization to 30 minutes reduces the duration of the protocol to approximately 4 hours in total (column 2). However, the number of PCR replicates increases compared to the control. As the number of PCR replicates increases, the diversity of the library also decreases and the corresponding coverage at approximately 20× decreases. Eliminating PCR from the 30 minute workflow (column 3) significantly reduces PCR replicates, increases diversity to levels above the control, and significantly shortens the workflow (2 hours versus 7.5 hours). . This example highlights that by eliminating PCR, the quality of hybridization capture reactions can be improved by minimizing template replication during PCR.


Example 8 - Comparison of blockers

with 0.2 mM of blockers BN001 and BN002; BX003 and BX007; BN007 and BN008; BN005 and BN006; or BN023, BN024, BN025 and BN026 (as described in Table 2A) in enhanced hybridization buffer. , hybridization and capture are performed as described in Example 1. "Unmodified" blockers BN001 and BN002 contain a thymine in the region of the blocker that corresponds to the index region of the adapter and a base complementary to the adapter in the region of the blocker that corresponds to the non-index region of the adapter. Blockers BN001 and BN002 do not contain modified bases and contain a 3'-ddC terminal group. Blockers BX003 and BX007 are modified versions of BN001 and BN002, each having two AP-dC-CE phosphoramidite (G-Clamp) substitutions in the region of the blocker that corresponds to the non-index region of the adapter. Blockers BN007 and BN008 have a universal base (deoxyinosine) in the region of the blocker that corresponds to the index region of the adapter, and a modification of BN001 and BN002 that has a BNA modified base in the region of the blocker that corresponds to the non-index region of the adapter. This is the version. Blockers BN023 and BN025 are modified versions of the region of BN001 that corresponds to the non-indexed region of the adapter and contain LNA modified bases, these blockers also contain a 3'-spacer C3 instead of 3'-ddC. Blockers BN024 and BN026 are modified versions of the region of BN002 that corresponds to the non-index region of the adapter and contain LNA modified bases; these blockers also contain a 3'-spacer C3 instead of 3'-ddC.

The resulting captured DNA is sequenced and the Padded Read Enrichment, a measure of the amount of on-target DNA captured, is calculated. The results are shown in Figure 10 and show that various modifications of "unmodified" blockers improve Padded Read Enrichment in hybridization assays.
Example 9 - Split Blocker Comparison

Hybridization and capture were performed as described in Example 1 using 0.2mM BN001 and BN002; or BN023, BN024, BN025 and BN026 (as described in Table 2A) in enhanced hybridization buffer. to be done. Corresponding adapters contain an 8 nucleotide index or a 10 nucleotide index. The resulting captured DNA is sequenced and Padded Read Enrichment is calculated. The results are shown in Figure 11 and show that by using "split blockers" (i.e., blockers that do not contain bases corresponding to the index region of the adapter), these blockers can be combined with various index sequences and index lengths. Indicates that it can be used with an adapter that has a These results indicate that split blockers can be used in situations where accommodating changes in index design may be disadvantageous.

All patents, patent applications and publications cited herein as well as electronically available materials (e.g., nucleotide sequence entries in GenBank and RefSeq and amino acid sequence entries in, e.g., SwissProt, PIR, PRF, PDB; and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. If there is a conflict between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples have been provided solely for clarity of understanding. No unnecessary limitations should be understood from them. The invention is not limited to the precise details shown and described, with variations obvious to those skilled in the art falling within the scope of the invention as defined by the claims.
In certain embodiments, for example, the following items are provided:
(Item 1)
A hybridization buffer comprising a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.
(Item 2)
The hybridization buffer according to item 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine.
(Item 3)
Hybridization buffer according to item 1 or item 2, comprising a crowding agent and a blocker.
(Item 4)
Hybridization buffer according to item 1 or item 2, comprising a crowding agent, human Cot-1 DNA, a blocker, a destabilizing agent, a buffer, a salt and a surfactant.
(Item 5)
The hybridization buffer according to any one of items 1 to 4, wherein the hybridization buffer comprises a buffer, and the buffer is a phosphate buffer.
(Item 6)
Hybridization buffer according to any one of items 1 to 5, wherein the hybridization buffer comprises a salt, and the salt is NaCl or sodium citrate.
(Item 7)
Hybridization buffer according to any one of items 1 to 6, wherein the hybridization buffer comprises a destabilizing agent, and the destabilizing agent is formamide or urea or a mixture thereof.
(Item 8)
Any one of items 1 to 7, wherein the hybridization buffer comprises a surfactant, and the surfactant is TWEEN® 20, TWEEN® 80 or sodium dodecyl sulfate (SDS). Hybridization buffer as described in .
(Item 9)
The hybridization buffer is
0.5% to 10% dextran sulfate,
0.05mg/mL to 0.5mg/mL human Cot-1 DNA,
1% to 15% (v/v) formamide,
40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ ,
0.1M to 4M NaCl, and
0.001% to 10% (v/v) TWEEN (registered trademark) 20
The hybridization buffer according to item 1, comprising:
(Item 10)
The hybridization buffer includes a blocker, the blocker comprising:
(a) Multiple modifications that increase T _m ;
(b) Bridged oligonucleotide; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA); tricyclic nucleic acid; peptide nucleic acid (PNA); ); a C5 modified pyrimidine base; a propynylpyrimidine; a morpholino; _a phosphoramidite; and a 5'-pyrene cap .
(c) an oligonucleotide, wherein said blocker binds to an adapter.
the adapter may include a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); The region has at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases, or at least 10 thymine bases. a thymine base; or an oligonucleotide comprising a universal base and at least one non-universal base; or
(d) two unconnected oligonucleotides, wherein the blocker is capable of binding at least a portion of an adapter, the adapter comprising a universal primer sequence and an index region or a unique molecular identifier (UMI ); the unconnected oligonucleotides contain bases that do not correspond to the index region and/or the UMI of the adapter;
Hybridization buffer according to any one of items 1 to 9, comprising at least one of:
(Item 11)
A method of preparing an enriched library, the method comprising: hybridization comprising a library member, a hybridization buffer according to any one of items 1 to 10, and a probe to form a hybridization mixture. A method comprising forming a mixture under conditions sufficient for said probe to hybridize to a region of interest within a library member, wherein said probe comprises a ligand.
(Item 12)
capturing the hybridized probe using a capturing means, and
eluting the captured library members from the capture means into an eluent containing enriched library members, wherein the DNA concentration of the eluent is within the range of 1.3 pM to 250 pM; process
The method according to item 11, comprising:
(Item 13)
The hybridization mixture comprises samples from at least two library preparations, wherein at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng or at least 500 ng of DNA from each library preparation is combined. The method according to item 11 or item 12.
(Item 14)
The method according to any one of items 11 to 13, wherein the DNA concentration of the hybridization mixture is in the range of 0.1 ng/μL to 120 ng/μL.
(Item 15)
Any one of items 11-14, wherein the method comprises sequencing the enriched library member, and the method does not include amplifying the library member prior to capturing the library member. The method described in.
(Item 16)
said sequencing step is carried out on a flow cell, said method comprises said enriched library member at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM and up to 100 pM; 16. The method of item 15, comprising loading the flow cell at a concentration of up to 200 pM, up to 250 pM or up to 300 pM.
(Item 17)
17. The method of item 16, comprising loading the enriched library member onto a flow cell using a direct flow cell loading jig.
(Item 18)
A kit comprising a hybridization buffer according to any one of items 1 to 10 and instructions for use.
(Item 19)
A blocker comprising an oligonucleotide, said blocker capable of binding to an adapter, said adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), said adapter The index region of the blocker and/or the region of the blocker capable of binding to the UMI comprises at least 3 thymine bases, at least 4 thymine bases, at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases. , at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases; or a universal base and at least one non-universal base.
(Item 20)
A blocker comprising two unconnected oligonucleotides, said blocker being capable of binding at least a portion of an adapter, said adapter comprising a universal primer sequence and an index region or unique molecular identifier (UMI). and wherein the unconnected oligonucleotide comprises a base that does not correspond to the index region and/or the UMI of the adapter.
(Item 21)
The universal primer sequences include P5, P7, P5', P7', V2. A14. METS, V2. B15. METS, V2. A14. Complementary strand of METS and V2. B15. The blocker according to item 19 or item 20, comprising at least one complementary strand of METS.
(Item 22)
22. The blocker of any one of items 19-21, wherein the blocker comprises a modification that increases the T _m of the blocker compared to the same blocker without the modification .
(Item 23)
DNA or RNA oligonucleotides modified such that the blocker captures low GC regions; bridge oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids. (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynylpyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. , the blocker according to any one of items 19 to 22.

Claims (17)

A hybridization buffer comprising a crowding agent, human Cot-1 DNA, a destabilizing agent, a salt, and a blocker , wherein the blocker is capable of binding to an adapter, the adapter comprising a universal primer sequence and an index region. or a unique molecular identifier (UMI); the index region of the adapter and/or the region of the blocker capable of binding to the UMI comprises at least three thymine bases, at least four thymine bases, A hybridization buffer comprising at least 5 thymine bases, at least 6 thymine bases, at least 7 thymine bases, at least 8 thymine bases, at least 9 thymine bases or at least 10 thymine bases. 2. The hybridization buffer of claim 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine. The hybridization buffer according to claim 1 or claim 2, comprising a crowding agent, human Cot-1 DNA, a blocker, a destabilizing agent, a buffer, a salt and a surfactant. The hybridization buffer according to any one of claims 1 to 3 , wherein the hybridization buffer comprises a buffer, and the buffer is a phosphate buffer. Hybridization buffer according to any one of claims 1 to 4 , wherein the salt is NaCl or sodium citrate. Hybridization buffer according to any one of claims 1 to 5 , wherein the destabilizing agent is formamide or urea or a mixture thereof. Any one of claims 1 to 6 , wherein the hybridization buffer contains a surfactant, and the surfactant is TWEEN (registered trademark) 20, TWEEN (registered trademark) 80, or sodium dodecyl sulfate (SDS). Hybridization buffer as described in Section. The hybridization buffer is
0.5% to 10% dextran sulfate,
0.05mg/mL to 0.5mg/mL human Cot-1 DNA,
1% to 15% (v/v) formamide,
40mM to 80mM KH ₂ PO ₄ -K ₂ HPO ₄ ,
0.1M to 4M NaCl, and 0.001% to 10% (v/v) TWEEN® 20
The hybridization buffer according to claim 1, comprising: The blocker is
(a) multiple modifications that increase T _m ; or
(b) Bridged oligonucleotide; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA); tricyclic nucleic acid; peptide nucleic acid (PNA); ); a C5 modified pyrimidine base; a propynylpyrimidine; a morpholino; _a phosphoramidite; and a 5'-pyrene cap .
Hybridization buffer according to any one of claims 1 to 8 , comprising at least one of: 10. A method of preparing an enriched library, the method comprising: a hybridization comprising library members, a hybridization buffer according to any one of claims 1 to 9 , and a probe to form a hybridization mixture. A method comprising forming a hybridization mixture under conditions sufficient for said probe to hybridize to a region of interest within a library member, wherein said probe comprises a ligand. capturing the hybridized probe using a capture means; and eluting the captured library member from the capture means into an eluent containing enriched library members, the steps comprising: 11. The method of claim 10 , comprising the step of: the eluent having a DNA concentration in the range of 1.3 pM to 250 pM. The hybridization mixture comprises samples from at least two library preparations, wherein at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng or at least 500 ng of DNA from each library preparation is combined. 12. The method according to claim 10 or claim 11 , wherein: The method according to any one of claims 10 to 12 , wherein the DNA concentration of the hybridization mixture is in the range of 0.1 ng/μL to 120 ng/μL. 14. The method of any of claims 10 to 13 , comprising the step of sequencing said enriched library members, said method not including the step of amplifying said library members before the step of capturing said library members. or the method described in paragraph 1. said sequencing step is carried out on a flow cell, said method comprises said enriched library member at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM or at least 100 pM and up to 100 pM; 15. The method of claim 14 , comprising loading the flow cell at a concentration of up to 200 pM, up to 250 pM or up to 300 pM. 16. The method of claim 15 , comprising loading the enriched library member onto a flow cell using a direct flow cell loading jig. A kit comprising a hybridization buffer according to any one of claims 1 to 9 and instructions for use.